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546003	Fully automated synthesis of (phospho)peptide arrays in microtiter plate wells provides efficient access to protein tyrosine kinase characterization	Background Synthetic peptides have played a useful role in studies of protein kinase substrates and interaction domains. Synthetic peptide arrays and libraries, in particular, have accelerated the process. Several factors have hindered or limited the applicability of various techniques, such as the need for deconvolution of combinatorial libraries, the inability or impracticality of achieving full automation using two-dimensional or pin solid phases, the lack of convenient interfacing with standard analytical platforms, or the difficulty of compartmentalization of a planar surface when contact between assay components needs to be avoided. This paper describes a process for synthesis of peptides and phosphopeptides on microtiter plate wells that overcomes previous limitations and demonstrates utility in determination of the epitope of an autophosphorylation site phospho-motif antibody and utility in substrate utilization assays of the protein tyrosine kinase, p60 c-src . Results The overall reproducibility of phospho-peptide synthesis and multiplexed EGF receptor (EGFR) autophosphorylation site (pY1173) antibody ELISA (9H2) was within 5.5 to 8.0%. Mass spectrometric analyses of the released (phospho)peptides showed homogeneous peaks of the expected molecular weights. An overlapping peptide array of the complete EGFR cytoplasmic sequence revealed a high redundancy of 9H2 reactive sites. The eight reactive phospopeptides were structurally related and interestingly, the most conserved antibody reactive peptide motif coincided with a subset of other known EGFR autophosphorylation and SH2 binding motifs and an EGFR optimal substrate motif. Finally, peptides based on known substrate specificities of c-src and related enzymes were synthesized in microtiter plate array format and were phosphorylated by c-Src with the predicted specificities. The level of phosphorylation was proportional to c-Src concentration with sensitivities below 0.1 Units of enzyme. Conclusions The ability of this method to interface with various robotics and instrumentation is highly flexible since the microtiter plate is an industry standard. It is highly scalable by increasing the surface area within the well or the number of wells and does not require specialized robotics. The microtiter plate array system is well suited to the study of protein kinase substrates, antigens, binding molecules, and inhibitors since these all can be quantitatively studied at a single uniform, reproducible interface.	Background Phosphorylation and dephosphorylation of proteins are major mechanisms mediating signal transduction throughout the cell and are intimately involved in the regulation of cell growth, physiology, differentiation, and death. Phosphorylation is accomplished by means of kinases which when stimulated by an afferent signal transmit the signal via phosphate transfer to the next site in a pathway. In some cases phosphoprotein-protein interactions take place that modulate signal transduction, e.g. by revealing previously sequestered phosphoacceptor sites in one or both of the interacting proteins, thus creating branch points in pathways. Critical questions exist regarding the identification of the true in vivo substrates of kinases, identification of phosphotyrosine interaction domains, and mapping the radiation of these protein interactions throughout extremely complex networks. Clearly new technologies capable of accelerating the processes for defining the interactions between kinases and their substrates and modulators would be of great value. Two highly productive approaches have been the determination of optimal substrate motifs favored by individual kinases, by various combinatorial peptide library approaches and, the use of antibodies to study phosphorylated peptide motifs (reviewed in [ 1 , 2 ]). Synthetic peptides have played a long and useful role in characterizing kinase substrate sequences, particularly for the ser/thr family, which is now seen to consist of a few distinct category types, basophilic, acidophilic and proline directed. Protein tyrosine kinases, on the other hand, are less well defined by their natural substrates but make more use of docking intermediaries to perform the task of substrate recognition. Nevertheless, optimal substrates have been found which can then aid in the search for the identity of natural or in vivo targets and inhibitors of the kinase [ 3 , 4 ]. While capable of assessing mixtures of very large numbers of random peptides, combinatorial methods require deconvolution strategies, which can be time-consuming, and technically demanding. A second search strategy for functional peptides employs arrays of spatially addressable peptides that can be tested in situ , accelerating the deconvolution process when the number of combinations is, or becomes more limited. Peptide arrays capable of displaying diverse functions including kinase substrate activity have been successfully produced by two methods: in situ synthesis on planar membranes or arrays of pins [ 5 - 10 ], or attachment of preformed peptides as performed in a variety of microarray printing procedures. While the existing synthetic methods are capable of producing large numbers of peptides in good purity, none are fully automatable. They require manual intervention between each synthesis cycle and thus are not totally automatable. Peptide synthesis in microtiter plate wells would allow the use of fully automated robotic handlers. Further, arrays of peptides produced in a microtiter plate format, which is an industry standard for numerous types of high throughput analytical procedures, could also be tested in automated multiplex fashion. We present data here demonstrating the applicability of an automated system for peptide design and synthesis in microtiter plates to the production of peptides and phosphopeptides. We further demonstrate the capability of these peptide arrays to be recognized correctly by specific phospho-motif antisera and to serve as kinase substrates. Results and discussion (Phospho)peptide synthesis method Previous work led to the development of a system for the automated synthesis of peptide arrays on the inner surfaces microtiter plate wells [ 11 - 13 ], (Figure 1 ). However, direct characterization of the synthesized peptides did not become realizable until the recent availability of modern high-sensitivity mass spectrometers. In this method polymethylpentene (TPX) microtiter plates are activated by oxidation with nitric acid, and made functional for peptide synthesis by condensation with poly(D-lysine) (n = 100), which serves as a free-floating support or polymeric handle containing an extended array of amino groups. Each lysine side-chain then serves as an initiation point for synthesis. The synthesized peptides are extended from the well surface by their attachment through their peptide carboxyl-termini, on a molecular tether of average estimated length of 50 lysine subunits. This simply assumes that each polylysine molecule is attached to the surface through one bond at its midpoint. It is expected that multiple attachment bonds between the surface and a single polylysine chain can also form but would be minimized by the 2000-fold molar excess of polylysine over attachment capacity used during the polylysine coating step. Model experiments have shown that the polylysine helical structure would be maintained in solution [ 14 , 15 ] for most, if not all cases. If multiple attachment bonds were formed then distal ends would prefer the helical form while the sequences between attachment points should also prefer the helical form up to the limits imposed by torsional constraints, and depending on the closeness of the attachment positions. Thus, the peptides are well positioned spatially, to interact with a variety of macromolecules such as antibodies and structures as large as a cell surface. The lengths and sequences of the peptides are programmable; the total elapsed cycle time to extend each peptide by one residue for all 96 wells is 1 hour. The process has been optimized with respect to activation conditions, length and composition of the polymeric tether and conditions for the handling and storage of the pre-diluted amino acid derivatives and condensing agents. Stabilities exceeding two years have been achieved for all reagents used. The capability of these peptides to be recognized by antibodies, leading to the identification of sequences and structures of the immunoreactive domains of viral proteins and biological response modifiers has been previously been demonstrated [ 11 - 13 ]. Reproducibility and quality of synthetic peptides To evaluate the fidelity and reproducibility of peptide syntheses and ability of the synthetic peptide arrays to serve as specific targets in sequence defined molecular affinity interactions, a model system was chosen to provide known test parameters. The EGF receptor system was selected since it provided commercially available monoclonal antibodies with documented specificity for an activation state associated autophosphorylation site (pY1173) and known sequence (NAEpYLRV). Testing was begun with the monoclonal antibody 9H2 produced against a peptide containing the NAEpYLRV sequence. An array of alternating NAEYLRV and NAE(pY)LRV peptides was prepared in a microtiter plate consisting of 12 8-well strips. Monoclonal 9H2 antibody ELISA was performed using three of the strips from the middle section of the plate. It was found that the antibody reacted strongly with the peptide wells containing phosphorylated tyrosine but not with the non-phosphorylated peptide wells or control wells without peptide (Figure 2 ). For the three strips ELISA means and (standard deviations) for the phosphotyrosine peptides were 3.62(0.20), 3.64(0.25), 3.67(030) and for the tyrosine peptides were 0.071(0.003), 0.068(0.007), 0.076(0.004). For wells containing no peptide, the values for all three strips were 0.063(0.0006). The coefficients of variation for all replicate sets ranged between 5% and 8%. For evaluation of the fidelity and authenticity of phosphorylated and non-phosphorylated peptide products wells containing NAEYLRV and NAEpYLRV were prepared as before except that a cleavable linker was added to the polylysine matrix before peptide synthesis. The synthesized peptides were then cleaved from the surface with TFA using conditions under which the protecting groups were removed from the amino acid side-chains but not from tyrosine phosphates. When the released peptide products were concentrated and analyzed by MALDI-TOF-TOF mass spectrometry it was found that both peptides yielded essentially monodisperse m/z of the predicted molecular weights, 1036.56 for NAEYLRV and 1170.62 for NAEpYLRV (Figure 3A and 3B , respectively; [bis(dimethylamino)phosphono]-tyrosine species shown). These data demonstrate high coupling efficiency at each step of synthesis and stability of the activated amino acids throughout the process. Redundancy of an autophosphorylation site antibody epitope in the EGFR cytoplasmic domain Since many of the twenty tyrosines found in the EGFR cytoplasmic domain are known to serve as substrate or SH2 binding site for other tyrosine kinases, it was decided to test the specificity of the 9H2 antibody. To examine this question, overlapping peptide arrays covering the transmembrane and cytoplasmic domains of the EGF receptor were prepared. One array contained only phosphotyrosine and the other only tyrosine. The arrays consisted of 92 peptides, each of which was 21 amino acids in length and overlapped by 15 amino acids. In the EGFR sequence arrayed, there are 20 unique occurrences of tyrosine-containing peptide sequences. In the array, each such sequence appears three to four times in progressively overlapping fashion. Clone 9H2 antibody showed high reactivity (from 9 to 57 times background) with eight out of the twenty phosphotyrosine-containing sequences (Figure 4 , also [see Additional file 1 ]). In general, each reactive peak was associated with three to four appearances of the identifiable tyrosine as its position moved progressively along the overlapping peptide sequences, suggesting consistent reliable synthesis throughout the synthetic process. None of the non-phosphorylated tyrosine-containing peptides showed any comparable reactivity with the monoclonal antibody 9H2 although a barely detectable level of antibody reactivity with all peptides containing tyrosine could be seen. There was no detectable reactivity against peptides not containing tyrosine [see Additional file 1 ]. Steric hindrance by the attachment matrix did not appear to be a significant problem in the recognition of reactive peptide sequences separated by just one, and two amino acids from the peptide carboxyl terminus ([see Additional file 1 ], array positions 81, 91) To assess the significance of these primary cross-reactivities a consensus table was constructed from 9H2 antibody reactive and non-reactive sequences (Figure 5 ). A strong preference for hydrophobic amino acids in the Y+1 position is readily apparent with leucine the most preferred appearing in 6 of 8 peptides, followed by isoleucine and valine, both appearing once. At the Y-1 position glutamic acid was the most preferred, appearing in 4 of 8 peptides, followed by arginine, asparagine, aspartic acid, and glutamine with one appearance each. Thus the preference appears to be primarily for glutamic acid but other hydrophilic amino acids were also accepted. There did not appear to be any discernable pattern of preference at the remaining peptide positions. The least common denominator among positive peptides therefore appears to be E/(R, N, D, Q)-p Y - L/(V, I), or E-pY-L in its predominant form. Peptide sequence # 84 [see Additional file 1 ] which lacked an acidic or hydrophilic amino acid at the pY-1 position was still strongly reactive demonstrating the strong contribution of a hydrophobic amino acid in the Y+1 position. However, since the peptide at position Y730 containing A(pY)V was not reactive ([see Additional file 1 ], Figure 5 ), 9H2 binding appears to involve more than the pY-hydrophobic sequence alone. Three of the 9H2 cross-reactive phosphotyrosines are clustered within the C-terminal end of the receptor (992–1173), are known autophosphorylation sites and are known to be recognized by proteins containing Group III SH2 binding domains [ 16 ] (e.g. p85, phospholipase C γ1 , the tyrosine phosphatases, Figure 5 ), that similarly recognize phosphotyrosines with hydrophobic amino acids at the Y+1 position. Since some of the 9H2 cross-reactive epitopes are not associated with phosphoacceptor activity it suggests that the phosphoacceptor site specificities are more stringently controlled than the 9H2 epitope or that phosphoacceptor activity simply has not been demonstrated yet. A similarity in the processes for recognition of specificity determinants within the deduced epitope of the 9H2 autophosphorylation site antibody and optimal substrate motif found for the EGF receptor [ 16 - 18 ], E EEE Y F ELV, may also exist. Twelve of the twenty tyrosines present in the EGFR cytoplasmic domain were not reactive with 9H2. None of the twelve conformed to the deduced 9H2-epitope motif, although six out of 12 were involved with other aspects of kinase signal transduction (Figure 5 ). As confirmation that this was not a result of failure to incorporate phosphotyrosine in the negative peptides a plate array containing all of the EGFR phosphotyrosine peptides was constructed and tested against a group of commercial phosphotyrosine antibodies prepared in various ways. These results are shown in Figure 6 . Antibodies 4G10, AB8076, and PT101L were prepared using immunogens which were chemically modified or haptenized and not known to be sequence restricted and all reacted strongly with all of the EGFR phosphopeptides. Antibody RDI-egfract-1 was prepared against the activated EGF receptor and known to be activation specific but not known to be active against linear peptides. These results add support to the specificity of the epitope deduced above and contribute additional useful information and reagents, namely confirmation of the pan-specific nature of the phosphotyrosine antibodies as used in the microtiter plate peptide array system Tyrosine kinase activity on synthetic peptide substrates in microtiter plate arrays To characterize the quantitative aspects of substrate phosphorylation by c-Src kinase, a panel of peptides based on known substrate specificities of c-src and related enzymes were synthesized in microtiter plate wells. Each peptide extended from the polylysine backbone by an ε-amino side-chain and by an additional C-terminal Cys unit. Thus, all of the peptides were equally available to the enzyme, with reduced steric hindrance and a uniform presentation. Subsequently, 90 μL of reaction buffer containing varying amounts of c-Src kinase were incubated for 20 min at 30°, according to the manufacturer's instructions. The wells were then washed with distilled water and assayed for the presence of phosphotyrosine by ELISA using a mixture of the broadly cross-reacting phosphotyrosine antibodies previously described in Figure 6 . The peptide substrates E E IYGEF F [ 17 ] (Src,1) and YIYGSFK [ 19 ] (Src,2) have been shown separately by somewhat different combinatorial methods to have relatively potent activity for protein tyrosine kinases and are shown to be reactive here as well (Figure 7 ). (Src, 1) was phosphorylated to a greater extent than (Src, 2) and showed no decrease of reactivity even at 0.1 Unit of enzyme, the lowest concentration used. The (Src, 1) variant peptides were chosen so that validation could be made by direct comparison with the highly oriented peptide chip system recently described [ 20 ]. The (Src, 1) variant peptide (-E) made by truncation of the N-terminus still showed good reactivity, although much lower than the longer (Src,1) peptide, and the (-Y) peptide in which tyrosine was exchanged for phenylalanine showed no reactivity as expected and required. The slight c-ABL tyrosine kinase substrate peptide IYAAPKKK [ 17 ] reactivity at the highest c-Src input and negative protein kinase A substrate peptide LRRASLGC [ 21 , 22 ] activity are consistent with the level of cross-reactivities expected between familial and nonfamilial kinases. Three of the peptide substrates showed dose-response characteristics conforming, by nonlinear regression, to a one site binding model for (-E), (Src, 2), and (ABL), with R 2 equal to 0.98, 0.99, and 0.92. Activity of the most reactive peptide, (Src, 1) was greater than expected based on quantity of enzyme used in previously published work [ 23 , 24 ] and was at least 30 times more reactive than its truncated form (-E) [ 20 ]. By extrapolation, c-Src activity would be detectable at concentrations as low as 0.01 Units of enzyme using the (Src, 1) substrate. Conclusions The method for production of synthetic peptide solid-phase arrays in microtiter plates described here is capable of making high quality peptides, as seen by mass spectrometry of the released unfractionated products. The peptide synthesis method is completely automated and has been greatly simplified by the use of standard automated liquid handlers and the use of activated amino acid solutions that may be prepared and stored in advance and added just once at the beginning of the synthetic process. In the present demonstration, 96 well microtiter plates were used; but, 384, 1536, or containers of any well density compatible with the solvents and liquid handler may be used. The solid phase peptide arrays produced on a polylysine backbone were found to be of very high density, provide very low levels of nonspecific binding and steric hindrance, and participate effectively in a variety of biochemical reactions. The strategy described here for the preparation of solid phase synthetic peptide arrays in microtiter plate wells for use in multiplexed assays offers many advantages in the study of protein kinases, particularly in a research environment. In a research environment combined cycles of hypothesis generation and testing, with assay flexibility, speed, quantitative accuracy and precision are of greater concern than in large scale screening applications of large numbers using limited, previously selected variables. The industry standard microtiter plate format ensures compatibility with a vast number of assay platforms and the polylysine backbone with its extended three-dimensional display provides a highly efficient, sterically unhindered, and extremely low background display of the peptide products. Using 96-member peptide arrays of 21-mers created in less than 24 hours, we have shown that the peptide array synthesis provided a highly reproducible model for a tyrosine peptide, EGFR Y1173 and its phosphorylated counterpart. Using a monoclonal antibody prepared against a synthetic peptide representation of the Y1173 EGF receptor autophosphorylation site, we have provided evidence that, unexpectedly, the deduced epitope, E/H L -pY-L/H B (where H L - is hydrophilic and H B is hydrophobic) is highly redundant within the cytoplasmic domain. Three of the eight antibody reactive sites have been previously identified as autophosphorylation sites and are recognized by Group III SH2 domain proteins (Figure 5 ) that have similar specificity patterns. Furthermore, the EGFR substrate sequence EEEEYFELV, derived by combinatorial peptide optimization [ 17 ], resembles the E-Y-(hydrophobic) motif found in these studies. Thus there is a consistent linkage between a subset of EGFR phosphotyrosine sequences recognized by the 9H2 antibody, a subset of sequences autophosphorylated by EGFR kinase, and EGFR autophosphorylation sites recognized by the Group III SH2 domain. There is a clear parallel between the 9H2 peptide epitope and the peptide substrate specificity of the EGFR catalytic activity [ 16 ]. Songyang has further suggested that the catalytic and SH2 domains of PTKs may have converged to recognize similar sequences. So, questions regarding which site (or sites) the antibody actually recognizes on stimulated EGF receptor molecules and what other parallels might exist between 9H2 binding, SH2 binding, and catalytic substrate selection become of interest. At the cell protein level, 9H2 is specific by Western blot for the stimulated EGF receptor. Furthermore, there are preliminary data (Saxinger, unpublished) suggesting that PDGF peptide sequences conforming to the 9H2 binding sequence of (hydrophilic)-pY-(hydrophobic) deduced from EGFR phosphotyrosine peptides, appear to be recognized differently by 9H2. While nine of the twenty-seven PDGFR phosphotyrosine sequences satisfied the (hydrophilic)-pY-(hydrophobic) sequence definition, and could be expected to be as reactive as those in EGFR, only one showed comparable reactivity. Thus, the binding determinants of 9H2 are more complex than the simple epitope deduced from Figure 5 . An intriguing possibility is that the 9H2 epitope may be a fairly simple one but that its appearance, or access to it has been limited or distorted in specific ways by spatially adjacent sequences or structures that have evolved to create opportunities for exploitation in biologically specific processes. We have also successfully demonstrated the use of microtiter plate peptide arrays in faithfully reproducing the known substrate phosphorylation specificities of c-Src protein kinase. In these studies a broadly reactive phosphotyrosine antibody ELISA detected phosphorylation. Although a mixture of antibodies that were not known to be sequence restricted was used, the possibility exists that some tyrosine-containing peptides could become phosphorylated and be recognized less well than others. Therefore in studies where the need for precise quantitation outweighs the convenience and safety considerations of ELISA, incorporation of radioisotopic phosphate would provide an alternative. The microtiter plate format with reactants bound to the well surface would provide a well-contained and safe vehicle for washing and subsequent measurement of radioactivity. Thus, the microtiter plate array system is well suited to the study of protein kinase substrates, antigens, related binding molecules, and inhibitors since these all can be quantitatively studied at a single uniform, reproducible interface. For applications requiring larger numbers of solid phase peptides, the synthetic process can easily be transferred to more powerful workstations such as the Biomek FX platform in which many plates with higher well densities can be synthesized simultaneously and conventional particle based substrates for peptide synthesis can be manipulated using filter plates or magnetic devices available for this system. Moreover, the current capacity of approximately 150–380 pMoles/well can be considered large enough for preparative or analytical applications coupled with mass spectrometric analyses, such as affinity-based proteomic screening for ligand-protein interactions. In this and other applications, such as assessment of protease activity where strict isolation of adjacent components is required, microtiter plate wells enjoy a significant mechanical advantage over two-dimensional spotting or other synthesis methods. Methods Materials Solvents and reagents for peptide synthesis were obtained as synthesizer grade from Applied Biosystems (Foster City, CA). Specifically, these were N-methylpyrollidone (NMP), 1 M N-Hydroxybenzotriazole (HOBT) in NMP, 1 M dicyclohexylcarbodiimide (DCC) in NMP, diisopropylethylamine (DIPEA), acetic anhydride, and trifluoroacetic acid (TFA). NMP from this vendor was consistently free of basic impurities and was stored over Molecular Sieve (4A) after opening. NMP solutions were stored at -20C and allowed to attain room temperature before opening. FMOC-protected α-amino acids were purchased from Penninsula Laboratories (San Carlos, CA) and side-chain substitutions were, Asn(Trt), Asp(OtBu), Cys(Acm) or Cys(Trt), Gln(Trt), Glu(OtBu), His(Trt), Lys(Boc), Ser(tBu), Thr(tBu) and Tyr(tBu). FMOC-Arg(Pbf), FMOC- [bis(dimethylamino)phosphono))-tyrosine] [ 25 ], and FMOC Rink amide (linker p- [(R,S)-a-[1-(9H-Fluoren-9-yl)-methoxyformamido]-2,4-dimethoxybenzyl]-phenoxyacetic acid) were purchased from NovaBiochem. Phenol, thioanisole, ethanedithiol (EDT), triisopropylsilane (TIPS), and carbonyldiimidazole (CDI) were obtained from Aldrich Chemical Co. (Milwaukee, WI). Poly(D-Lys•HBr) (dp 100) and Poly(L-Lys•HBr) (dp 100) were obtained from Sigma-Aldrich (St. Louis, MO). TPX (polymethylpentene) 8-well strips, non-sterile, non-tissue culture treated, were obtained from Costar on special order (Cambridge, MA). (Phospho)Peptide synthesis Automated preparation of solid phase synthetic peptide arrays in microtiter plates was performed as described previously [ 13 ] (Saxinger, US Patent 6031074, Feb. 29, 2000). Carboxyl functional groups are formed on the hydrocarbon surface of Costar TPX microtiter plate strip wells by oxidation with 70% nitric acid for two hours at 65°C or for two weeks at room temperature. Polylysine chains are then attached to the surface by condensation using 0.05 M carbonyldiimidazole in NMP for 30' at 20°C. Poly(L-Lys•Hbr) (PLL) or poly(D-Lys•Hbr) (PDL) (1 mg/ml in 90% NMP-10% water) was neutralized with DIPEA and reacted with the CDI-activated surface for 1 hour at 20°C and followed by 4°C overnight. Plates were rinsed twice with water and twice with methanol, air-dried, wrapped in plastic cling-wrap and stored at room temperature. Peptide synthesis next takes place by the sequential addition of Nα-FMOC amino acids activated with DCC/HOBT as in conventional peptide synthesis with the growing peptide chain covalently attached to the polylysine chain through its carboxyl terminus. Stock solutions of FMOC amino acids were prepared in advance by dissolving 6 mmoles of each in 12 mL of NMP containing a 10% molar excess of HOBT, and stored at -20°C. The solutions could then be used repetitively for at least three months. Prior to peptide synthesis, the required amounts of amino acid were diluted to 0.1 M in NMP, mixed with an equimolar volume of 0.1 M DCC in NMP and allowed to react for thirty min in 2 mL cryovials. The 20 mixtures were then placed in a 24 tube rack on the Biomek 1000 workstation tablet for use during subsequent peptide synthesis cycles. Synthesis automation is achieved through software that receives input designating the peptide desired in each of the 96 microtiter plate wells and creates output files to indicate the quantity of reagents needed and a set of Beckman Biomek 1000 arrays.bio files, each member of the set directing the distribution of amino acids in one of the sequential chain extension cycles. An automated repetitive set of reagent transfers and washing steps is then recycled using a new arrays.bio file for each cyle until the peptide synthesis is complete. After synthesis the peptides are usually capped by reaction with acetic anhydride (1 mL in 10 mL of NMP containing 0.1 mL of DIPEA per plate) before side-chain deprotection and testing, in multiplex fashion, for reactivity. Side chain protecting groups were generally removed with: 10 mL of TFA +0.75 g of crystalline phenol + 0.5 mL of purified water + 0.5 mL of thioanisole +0.25 mL of ethanedithiol per plate, incubated for three hours in a sealed container, washed with ether, air-dried in a chemical fume hood, and stored at -20°C in plastic wraps. For peptides not containing Met, Trp, or Cys, TFA containing TIPS and H 2 O was used (95%, 2.5%, and 2.5%, respectively). Peptide synthesis capacity of microtiter plate wells TPX microtiter plates were oxidized at either room temperature or 60°C and reacted with polylysine. Peptide quantities were estimated by two different methods. In the first, the polylysine amino groups were modified with the cleavable linker HMPB [4-(4-Hydroxymethyl-3-methoxyphenoxy)-butyric acid] and FMOC-L-Cys(Acm) was coupled to the HMPB-substituted support as described above. Terminal amino groups were deprotected with 20% piperidine in NMP, washed with NMP and derivatized with dabsyl chloride. The dabsyl-amino acid derivative was released with 95% TFA, harvested from multiple wells by serial transfer, and measured by spectrophotometric scanning in a Beckman DU65 using an extinction coefficient of Dabsyl Chloride = 6.5 × 10 4 in TFA, λ max = 495). In the second method, FMOC-L-Ala was coupled directly to the polylysine supports. The FMOC protecting group was released in 20% piperidine, harvested from multiple wells by serial transfer, and quantitated by fluorimetric scanning in a Perkin Elmer LS50B using E(301) = 7800 (1 mM/ml). Values for spectral constants and coefficients were determined from standards dissolved in the cleavage solvent. The solid phase capacity was 380 pMoles/well and the releasable capacity was 150 pMoles/well. Mass Spectrometry of synthetic array peptides A polylysine coated TPX microtiter plate was modified by preliminary reaction with FMOC-Rink Amide linker (Novabiochem) to allow peptide cleavage after synthesis using standard DCC/HOBT coupling conditions, as described above. All peptides were initiated with C-terminal Cys(Acm) to provide a constant initiation and cleavage environment. Peptides were released and deprotected by incubation for 3 hours at room temperature in 100 μL of TFA containing water, ethanedithiol and phenol, or TFA containing water and triisopropylsilane. An additional 10 uL of water and 16 hours of incubation time are required for deprotection of [bis(dimethylamino)phosphono))-tyrosine] [ 25 ]. Solutions of released peptides were concentrated by vacuum centrifugation and 0.25 μL of sample was co-crystallized with 0.25 μL of α-cyano-4-hydroxycinnamic acid in 50% ACN, 1% trifluoroacetic acid and spotted directly on a stainless steel matrix-assisted laser desorption ionization (MALDI) plate. Mass spectra were acquired using an Applied Biosystems 4700 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Foster City, CA). MALDI mass spectra were externally calibrated (<20 ppm) using a standard peptide mixture. Antibody recognition of microtiter plate arrayed peptides Goat anti-mouse IgG (phosphatase conjugated) was purchased from Kirkegaard & Perry (Gaithersburg, MD) and Upstate Biotechnology (Lake Placid, NY). Murine monoclonal antibody 9H2, purchased from Upstate Biotechnology was prepared against a synthetic peptide containing the EGFR autophosphorylation sequence at Y1173 and was certified by the manufacturer to be specific for EGF-stimulated A431 cells by Western blot. Murine antisera prepared against phosphotyrosine were 4G10 (Upstate), ab8076 (Abcam, Ltd.), and PT01L (Oncogene Research Products). Antiserum RDI-egfract-1 was prepared against isolated tyrosine phosphorylated (activated) EGF receptor from EGF-challenged murine L-cells (Research Diagnostics, Inc.). All sera were used according to the manufacturer's recommendations and assayed by indirect ELISA. The extent of reaction was determined using a phosphatase assay kit from Kirkegaard & Perry. Kinase assay Peptide array substrate evaluations were performed using p60 c-src protein-tyrosine kinase, Cat# PK03 (Oncogene Research Products) according to the instructions in the manufacturer's insert. In sequence, all wells received 30 μL of Kinase Assay Buffer (0.05 M HEPES, pH 7.5 + 0.1 mM EDTA + 0.015% BRIJ 35), 30 μL of appropriately diluted p60 c-src in Kinase Dilution Buffer (0.1 mg/mL BSA + 0.2% β-mercaptoethanol), 30 μL of ATP mix (0.03 M MgCl 2 +0.15 mM ATP) in Kinase Assay Buffer. Plates were incubated at 30°C for 30 min, rinsed with distilled water and assayed for the presence of phosphotyrosine as described above. Each unit of p60 c-src enzyme catalyzes the incorporation of one pMole of phosphate into tyrosyl residues. List of abbreviations used (9H2): antibody prepared against a synthetic peptide containing the EGFR autophosphorylation site at Y1173 carbonyldiimidazole (CDI) dicyclohexylcarbodiimide (DCC) diisopropylethylamine (DIPEA) EGF receptor (EGFR) ethanedithiol (EDT), N-Hydroxybenzotriazole (HOBT) N-methylpyrollidone (NMP) phosphotyrosine kinase (PTK) Polymethylpentene (TPX) trifluoroacetic acid (TFA) triisopropylsilane (TIPS), Amino acid and polypeptide abbreviations were in accordance with IUPAC-IUB recommendations. Authors' contributions WCS designed the strategy for peptide synthesis, participated in the design of the strategy for peptide synthesis validation, carried out the peptide synthesis, testing, functional analyses, and drafted the manuscript. TPC participated in the design of the strategy for peptide synthesis validation, carried out the mass spectrometry analyses and interpreted the ms results. DJG participated in the design of the strategy for peptide synthesis validation. TDV participated in the design of the strategy for peptide synthesis validation and coordinated the mass spectrometry analyses. Supplementary Material Additional File 1 EGRF (pY) peptide scanning array Vs 9H2 Antibody ELISA. EGF receptor phosphotyrosine and tyrosine overlapping peptide array sequences and ELISA test results for each peptide array. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546003.xml
516768	Most of the extant mtDNA boundaries in South and Southwest Asia were likely shaped during the initial settlement of Eurasia by anatomically modern humans	Background Recent advances in the understanding of the maternal and paternal heritage of south and southwest Asian populations have highlighted their role in the colonization of Eurasia by anatomically modern humans. Further understanding requires a deeper insight into the topology of the branches of the Indian mtDNA phylogenetic tree, which should be contextualized within the phylogeography of the neighboring regional mtDNA variation. Accordingly, we have analyzed mtDNA control and coding region variation in 796 Indian (including both tribal and caste populations from different parts of India) and 436 Iranian mtDNAs. The results were integrated and analyzed together with published data from South, Southeast Asia and West Eurasia. Results Four new Indian-specific haplogroup M sub-clades were defined. These, in combination with two previously described haplogroups, encompass approximately one third of the haplogroup M mtDNAs in India. Their phylogeography and spread among different linguistic phyla and social strata was investigated in detail. Furthermore, the analysis of the Iranian mtDNA pool revealed patterns of limited reciprocal gene flow between Iran and the Indian sub-continent and allowed the identification of different assemblies of shared mtDNA sub-clades. Conclusions Since the initial peopling of South and West Asia by anatomically modern humans, when this region may well have provided the initial settlers who colonized much of the rest of Eurasia, the gene flow in and out of India of the maternally transmitted mtDNA has been surprisingly limited. Specifically, our analysis of the mtDNA haplogroups, which are shared between Indian and Iranian populations and exhibit coalescence ages corresponding to around the early Upper Paleolithic, indicates that they are present in India largely as Indian-specific sub-lineages. In contrast, other ancient Indian-specific variants of M and R are very rare outside the sub-continent.	Background Two mtDNA macro-haplogroups (M and N) that arose from the African haplogroup L3 encompass virtually all mtDNAs outside Africa [ 1 - 4 ]. The phylogenetic node N (including R) has spread its branches all over Eurasia, in contrast to haplogroup M, which is found in Eastern Eurasia but is virtually absent in Europe. The numerous branches of N are, however, generally segregated to either the eastern (e.g. A, B [ 5 ], Y [ 6 ], R9 [ 7 ] or western (e.g. N1 [ 8 , 9 ], N2 (comprising of W and its sister-clade identified by [ 10 ]), TJ, HV, U [ 11 ]) Eurasian-specific pools. The majority of Indian mtDNAs belong to macro-haplogroup M [ 8 , 12 - 21 ]. While the topology of the M sub-haplogroups that are common in mainland East Asia (M7, M8 (including C, Z), M9 (including E), D, G [ 7 , 22 , 23 ]) and in Africa (M1 [ 24 ]) is established in detail, the internal haplogroup structure of M in India has remained largely undefined. We have previously demonstrated that transitions at nps. 477G, 1780, 8502 and 16319 designate Indian-specific haplogroup M2, the most frequent M clade in India [ 15 ]. Another Indian-specific M clade supported by HVS-I variation as well as coding region markers, is M6 [ 15 ]. Haplogroups M3, M4 and M5 have been discriminated preliminarily by their characteristic HVS-I mutations [ 19 ], but since their defining positions, 16126, 16311, and 16129, respectively, are phylogenetically unstable [ 25 , 26 ], it is unlikely that the proposed haplogroups are monophyletic. Most numerous sub-groups of macro-haplogroup N in India are the Indian-specific variants of the phylogenetic node R including haplogroups R5, R6, U2(a, b, c) [ 8 , 13 , 27 ]. The overwhelming majority of the Iranian mtDNAs have been shown to lie in the West Eurasian domain of the global human mtDNA pool [ 27 , 28 ]. Here we focus on the analysis of mtDNA lineages that are shared between Indians and Iranians and bear signals of pre-Holocene expansion in the region. India congregates four linguistic domains (Indo-European, Dravidic, Austro-Asiatic and Tibeto-Burman) that occupy non-random spheres of the geographic distribution of its populations. The majority of the recent studies based on mtDNA variation have, in contrast to some [ 21 ], provided evidence that linguistic groups of India do not represent genetically homogeneous units and are not, therefore, traceable to different immigration waves from distinct sources [ 8 , 13 , 19 ]. The complexity that arises in defining populations and groups of populations in India based on genetic and cultural criteria has been recently demonstrated in South Indian tribal and caste populations. The combined data from mtDNA, Y-chromosome and autosomal genes indicated that the tribes and castes derive largely from the same genetic heritage of Late Pleistocene southern and southwestern Asians, and have received limited gene flow from external sources since the Holocene [ 15 ]. Similar results were obtained by Cordaux et al. [ 29 ], who demonstrated that caste and tribal groups exhibit similar levels of molecular variance. However, genetic distances indicated that the Tibeto-Burman speaking tribal populations (from eastern India) were more closely related to East Asians than to other Indians [ 29 ]. This is consistent with an earlier suggestion placing the origin of these tribal groups east of India – in Tibet and Myanmar [ 30 ]. In this study, we have analyzed the mtDNA variation in a sample of 796 Indians and 436 Iranians (Table 1 ), and combined the results with previously published data from the same geographic area. We also compared the mtDNA variation in India and Iran with that of Europe, China, and Thailand. The overall aim was to improve our understanding of the origins and composition of the Indian and Iranian gene pools and to determine the nature and the extent of gene flow between these regions. Through the analyses of the genetic variation of extant Southwest and South Asian populations we took an endeavor to envisage the exodus of anatomically modern humans from Africa. Table 1 Characteristics of the Indian and Iranian population samples whose mtDNA variation has been determined in the course of this study. INDIA State / Region Socio-cultural affiliation Linguistic affiliation Population Code n N West Bengal Caste Indo-European Mixed caste people Ben 50 80 Million Uttar Pradesh Tribe Indo-European Bhoksa Bho 5 32,000 Kerala Caste Indo-European Mixed caste people from Cochin Co 55 600,000 Kerala - a Indo-European Cochin Jews CoJ 45 5,000 (in Israel) Gujarat Caste Indo-European Mixed caste people Guj 53 50 Million Himachal Tribe Tibeto-Burman Kanet Kan 37 33,000 Maharashtra Caste Indo-European Konkanastha Brahmin Kon 58 N/A West Bengal Caste Indo-European Kurmi Kur 55 N/A West Bengal Tribe Austro-Asiatic Lodha Lod 56 59,000 Sri Lanka Caste Dravidic Moor Mo 50 3.4 Million Maharashtra - a Indo-European Parsi Par 55 76,000 Punjab Caste Indo-European Mixed caste people Pun 109 24 Million Rajasthan Caste Indo-European Rajput Raj 35 5 Million (in Rajasthan) Sri Lanka Caste Indo-European Sinhalese Sin 82 14.6 Million Uttar Pradesh Tribe Indo-European Tharu Tha 26 96,000 (in India) Uttar Pradesh Caste Indo-European Uttar Pradesh Brahmin UPb 25 2.5 Million total 796 IRAN northwest 226 southwest 138 northeast 30 southeast 6 central 36 total Indo-European Iranian IR 436 ca. 68 Million a For some analyzes these populations where grouped with the caste populations. (see Table 1 in the Supplementary Material for the complete set of studied populations including those whose mtDNA variation was previously published) Results and Discussion Geographic distribution of macro-clades M and N in India We found haplogroup M ubiquitous at almost 58% among the caste, and 72% among the tribal populations (Table 2 ), which is largely consistent with previous reports [ 8 , 12 - 21 ]. Our results indicate that the frequency distribution of haplogroup M varies across different Indian regions by a significant cline towards the south and the east (see Figure 4 for Spatial Autocorrelation Analysis (SAA) p < 0.05). The variation among caste populations climbs from approximately 40% in Gujarat and (Indian-) Punjab to 65% in the southern states, and peaks at over 70% in West Bengal (Table 2 ). We observed a similar geographic pattern among tribal populations, where the frequency varied from just over 50% in the northern states of Punjab and Himachal Pradesh, increased to 70%–80% in the southern states, and peaked at 86% in West Bengal (Table 8, see Additional file 3 ). Figure 4 Spatial Autocorrelation Analyses Correlograms of different haplogroups and haplogroup groupings frequencies in South Asia. In the case of haplogroup M3a some datasets had to be excluded because lack of resolution (see footenote for Table 3. for detailes). There was no significant cline in the frequencies of haplogroup R2. In the case of haplogroups M6a and M6b only the potential cline along the Bay of Bengal is investigated. Therefor the number of distance classes is reduced to three. Table 2 Geographic, linguistic and socio-cultural distribution of major Indian-specific mtDNA haplogroups HAPLOGROUP FREQUENCY (95% CR FOR PROPORTION) n M b M2 M3a M4a M6 M18 M25 R c R5 R6 U2i d R2; U7; W EE e WE f India 2544 g 65 (63.1–66.8) 6.3 (5.4–7.3) 2.2 (1.7–2.9) 0.6 (.4–1) 2.9 (2.3–3.6) 2.1 (1.6–2.8) 0.9 (.6–1.4) 11.4 (10.2–12.7) 2.2 (1.7–2.8) 1.3 (.9–1.8) 5.3 (4.5–6.2) 3.4 (2.7–4.2) 10.5 (9.4–11.8) 12.1 (10.9–13.4) Iran 436 5.3 (3.6–7.8) 0 (0–.8) 0.5 (.1–1.6) 0 (0–.8) 0 (0–.8) 0.2 (.1–1.3) 0 (0–.8) 3.2 (1.9–5.3) 0.9 (.4–2.3) 0 (0–.8) 0.2 (.1–1.3) 13.3 (10.4–16.8) 3.9 (2.5–6.2) 90.8 (87.7–93.2) Socio-cultural affiliation (Indian data only) Caste population 1204 57.8 (54.9–60.5) 5.1 (4–6.5) 4.2 (3.2–5.5) 1.1 (.6–1.8) 3 (2.2–4.1) 1.4 (.9–2.2) 1.4 (.9–2.2) 14.1 (12.3–16.2) 3.3 (2.5–4.5) 1.7 (1.1–2.5) 7.8 (6.4–9.5) 4.8 (3.7–6.2) 4 (3–5.2) 17.1 (15.1–19.3) Tribal population 1332 71.5 (69–73.8) 7.5 (6.2–9) 0.5 (.2–1) 0.2 (0–.5) 2.7 (2–3.7) 2.7 (2–3.7) 0.5 (.2–1) 8.9 (7.5–10.6) 1.2 (.7–1.9) 0.9 (.5–1.6) 3 (2.2–4.1) 2 (1.4–2.9) 16.5 (14.6–18.6) 7.5 (6.2–9) Brahmins & Kshatriyas 313 60.1 (54.5–65.3) 1.9 (.9–4.1) 5.1 (3.2–8.1) 1 (.3–2.8) 0.6 (.2–2.3) 1 (.3–2.8) 2.2 (1.1–4.5) 14.7 (11.2–19.1) 4.5 (2.7–7.4) 1.3 (.5–3.2) 6.4 (4.2–9.7) 6.1 (3.9–9.3) 3.5 (2–6.2) 17.6 (13.8–22.2) Other Castes h 517 61.5 (57.2–65.6) 6.2 (4.4–8.6) 4.6 (3.1–6.8) 1.2 (.5–2.5) 2.9 (1.8–4.7) 2.1 (1.2–3.8) 0.8 (.3–2) 12.6 (10–15.7) 2.3 (1.3–4) 1.5 (.8–3) 7.4 (5.4–9.9) 3.9 (2.5–5.9) 1.7 (.1–1.4) 16.6 (1.2–3.8) Language groups of India Indo-European 963 57.5 (54.4–60.6) 3.8 (2.8–5.3) 4.5 (3.3–6) 1.0 (.6–1.9) 2.7 (1.9–3.9) 1.1 (.6–2) 1.6 (1–2.6) 7.8 (6.3–9.7) 2.5 (1.7–3.7) 1.6 (1–2.6) 7.1 (5.6–8.9) 6.9 (5.4–8.6) 4.0 (3–5.5) 19.9 (17.5–22.6) Dravidic 1063 70.0 (67.2–72.7) 10.8 (9.1–12.8) 1.2 (.7–2.1) 0.5 (.2–1.1) 4.0 (2.9–5.3) 1.4 (.9–2.3) 0.5 (.2–1.1) 8.9 (7.4–10.8) 2.7 (1.9–3.9) 1.4 (.9–2.3) 5.9 (4.7–7.5) 1.2 (.7–2.1) 3.0 (2.1–4.2) 8.8 (7.3–10.7) Tibeto-Burman 249 59.8 (53.6–65.7) 0 (0–1.5) 0.4 (.1–2.2) 0 (0–1.5) 1.6 (.7–4) 1.2 (.4–3.5) 1.2 (.4–3.5) 0.8 (.2–2.9) 1.2 (.4–3.5) 0 (0–1.5) 0.8 (.2–2.9) 2.0 (.9–4.6) 61.4 (55.3–67.3) 3.2 (1.7–6.2) Austro-Asiatic (Mundari) 90 86.7 (78.1–92.2) 2.2 (.7–7.7) 0 (0–4) 0 (0–4) 0 (0–4) 26.7 (18.6–36.7) 0 (0–4) 4.4 (1.8–10.9) 0 (0–4) 1.1 (.3–6) 1.1 (.3–6) 1.1 (.3–6) 0 (0–4) 1.1 (.3–6) Indo-European i 250 66.4 (60.3–72) 8.0 (5.3–12) 0.4 (.1–2.2) 0.8 (.2–2.8) 4.8 (2.8–8.2) 1.6 (.6–4) 0.4 (.1–2.2) 8.8 (5.9–13) 2.0 (.9–4.6) 1.6 (.6–4) 6.4 (4–10.1) 4.0 (2.2–7.2) 1.2 (.4–3.5) 13.6 (9.9–18.4) Indo-European j 713 54.4 (50.7–58) 2.4 (1.5–3.8) 5.9 (4.4–7.9) 1.1 (.6–2.2) 2.0 (1.2–3.3) 1.0 (.5–2) 2.0 (1.2–3.3) 7.4 (5.7–9.6) 2.7 (1.7–4.1) 1.5 (.9–2.7) 7.3 (5.6–9.4) 7.9 (6.1–10.1) 5.0 (3.7–6.9) 22.2 (19.3–25.4) Indian States and Bangladesh (excluding tribal populations) k Andhra Pradesh 245 65.3 (59.1–71.0) 7.7 (5–11.8) 2.8 (1.4–5.7) 1.6 (.6–4.1) 3.3 (1.7–6.4) 4.1 (2.3–7.4) 0 (0–1.5) 18 (13.7–23.2) 4.1 (2.3–7.4) 1.2 (.4–3.5) 5.3 (3.2–8.9) 2.4 (1.1–5.2) 1.2 (.4–3.5) 8.9 (6–13.2) Bangladesh 30 66.67 (48.6–80.8) 9.96 (3.61–25.7) 0 (.08–11.2) 0 (.08–11.2) 3.29 (.78–16.6) 3.29 (.78–16.6) 0 (.08–11.2) 6.67 (2.04–21.4) 0 (.08–11.2) 0 (.08–11.2) 6.67 (2.04–21.4) 3.29 (.78–16.6) 9.96 (3.61–25.7) 6.67 (2.04–21.4) Gujarat 57 40.4 (28.6–53.4) 5.3 (1.9–14.4) 7 (2.9–16.7) 1.8 (.4–9.2) 1.8 (.4–9.2) 1.8 (.4–9.2) 0 (0–6.2) 17.5 (9.9–29.4) 5.3 (1.9–14.4) 1.8 (.4–9.2) 1.8 (.4–9.2) 17.5 (9.9–29.4) 5.3 (1.9–14.4) 33.3 (22.5–46.3) Karnataka 47 h 55.3 (41.2–68.6) 2.1 (.5–11.1) 2.1 (.5–11.1) 0 (.1–7.4) 0 (.1–7.4) 0 (.1–7.4) 0 (.1–7.4) 19.1 (10.5–32.6) 6.4 (2.3–17.2) 0 (.1–7.4) 10.6 (4.7–22.7) 6.4 (2.3–17.2) 0 (.1–7.4) 14.9 (7.5–27.8) Kashmir 19 26.3 (11.9–49.1) 0 (.1–16.8) 0 (.1–16.8) 5.3 (1.2–24.9) 10.5 (3.2–31.7) 0 (.1–16.8) 0 (.1–16.8) 10.5 (3.2–31.7) 0 (.1–16.8) 5.3 (1.2–24.9) 10.5 (3.2–31.7) 5.3 (1.2–24.9) 21.1 (8.7–43.7) 31.6 (15.4–54.3) Kerala 100 54 (44.2–63.5) 4 (1.6–9.8) 0 (0–3.6) 0 (0–3.6) 0 (0–3.6) 0 (0–3.6) 5 (2.2–11.2) 25 (17.6–34.3) 9 (4.9–16.2) 1 (.2–5.4) 7 (3.5–13.8) 1 (.2–5.4) 4 (1.6–9.8) 11 (6.3–18.7) Maharashtra 117 65 (55.9–73) 3.4 (1.4–8.5) 11.1 (6.6–18.1) 0.9 (.2–4.6) 0 (0–3.1) 0 (0–3.1) 5.1 (2.4–10.7) 6.8 (3.5–12.9) 0.9 (.2–4.6) 0.9 (.2–4.6) 4.3 (1.9–9.6) 4.3 (1.9–9.6) 5.1 (2.4–10.7) 23.1 (16.4–31.5) Punjab 150 36 (28.8–44) 0.7 (.2–3.6) 4.7 (2.3–9.3) 1.3 (.4–4.7) 0.7 (.2–3.6) 0 (0–2.4) 2.7 (1.1–6.6) 11.3 (7.2–17.4) 0.7 (.2–3.6) 0.7 (.2–3.6) 7.3 (4.2–12.7) 14 (9.4–20.5) 5.3 (2.8–10.2) 42.7 (35–50.7) Rajasthan 36 69.4 (53–82) 0 (.1–9.5) 13.9 (6.2–28.8) 0 (.1–9.5) 0 (.1–9.5) 5.6 (1.7–18.2) 0 (.1–9.5) 22.2 (11.8–38.2) 5.6 (1.7–18.2) 2.8 (.7–14.2) 2.8 (.7–14.2) 5.6 (1.7–18.2) 0 (.1–9.5) 2.8 (.7–14.2) Sri Lanka 132 57.6 (49–65.7) 6.8 (3.7–12.5) 1.5 (.5–5.3) 1.5 (.5–5.3) 1.5 (.5–5.3) 0.8 (.2–4.1) 0 (0–2.7) 12.9 (8.2–19.7) 0.8 (.2–4.1) 3 (1.2–7.5) 12.1 (7.6–18.8) 2.3 (.8–6.5) 2.3 (.8–6.5) 15.2 (10–22.3) Tamil Nadu 51 64.7 (50.9–76.4) 5.9 (2.1–15.9) 3.9 (1.2–13.2) 2 (.5–10.3) 2 (.5–10.3) 2 (.5–10.3) 0 (0–6.8) 11.8 (5.6–23.4) 0 (0–6.8) 3.9 (1.2–13.2) 2 (.5–10.3) 3.9 (1.2–13.2) 23.5 (14–36.8) 7.8 (3.2–18.5) Uttar Pradesh 98 52 (42.2–61.7) 1 (.2–5.5) 8.2 (4.2–15.3) 1 (.2–5.5) 0 (0–3.7) 2 (.6–7.1) 1 (.2–5.5) 11.2 (6.4–19) 3.1 (1.1–8.6) 2 (.6–7.1) 15.3 (9.5–23.8) 3.1 (1.1–8.6) 4.1 (1.7–10) 13.3 (8–21.4) West Bengal 106 71.7 (62.5–79.4) 3.8 (1.5–9.3) 1.9 (.6–6.6) 0 (0–3.4) 5.7 (2.7–11.8) 0 (0–3.4) 0.9 (.2–5.1) 12.3 (7.3–19.9) 6.6 (3.3–13) 2.8 (1–8) 6.6 (3.3–13) 0.9 (.2–5.1) 0.9 (.2–5.1) 7.5 (3.9–14.2) a a See Tables 8–13 (additional files 3–7, 9) for frequencies of other haplogroups including breakdown of R2, U7 and W b Includes all haplogroup M sub-haplogroups c Includes sub-haplogroups R2, R5, R6 and R10 d Composite of haplogroups U2a, U2b and U2c [27] e Composite of haplogroups A, B, D, G, M7, M8 (including C and Z), M9 (including E), M10, N9 (including Y), R9 (including F) f Composite of haplogroups HV, TJ, X, N1, N2 and U (except U2i) g Caste and tribal populations plus 8 Siddi [49] who belong to neither groupings h Datasets where caste affiliation was not determined (undefined/mixed caste population), Scheduled Caste populations (e.g. the Mukri, see text for explanation) and data from [3] (dataset of 27 haplogroup M samples) are excluded. i Indo-European speakers of southern India (Tamil Nadu, Kerala, Karnataka, Andhra Pradesh and Sri Lanka) j Indo-European speakers of northern India (States other than listed in the previous footnote) k The sum of the presented caste populations sample sizes is different from that given in the third row of the current table because of the addition of the Bangladeshi (n = 30) and extraction of the Mukri (n = 42), Orissa (n = 2) and Bihar (n = 2) data. With the exception of the diverse set of largely Indian-specific R lineages, the most frequent mtDNA haplogroup in India that derives from the phylogenetic node N is haplogroup W [ 13 ]. The frequency peak of haplogroup W is 5% in the northwestern states – Gujarat, Punjab and Kashmir. Elsewhere in India its frequency is very low (from 0 to 0.9%) (Table 2 ) forming a significant spatial cline (Figure 4 ). At 15% among the caste and 8% among the tribal populations haplogroup U is the most frequent sub-clade of R in India (Table 12, see Additional file 7 ). Approximately one half of the U mtDNAs in India belong to the Indian-specific branches of haplogroup U2 (U2i: U2a, U2b and U2c) [ 13 , 27 ] (Table 2 ). They are present throughout India without a clear geographical cline (Figure 2 , panel U2i, SAA p > 0.05). However, the spread of another subset of U, haplogroup U7 [ 13 ], is similar to that of haplogroup W, peaking at 12% and 9% in Gujarat and Punjab, respectively (Table 11, see Additional file 6 ). The frequency of U7 is also high in neighboring Pakistan (6%) and particularly in Iran (9%) (Table 9, see Additional file 4 ). Figure 2 The spatial distribution of Indian-specific mtDNA haplogroups (U2, M4a, M18 and M25) and their sub-haplogroups. In the case of haplogroup M25 some datasets had to be excluded because the discrimination between M* and the respective subgroup was not possible on the basis of the HVS-I data alone (see footnote of Table 3). For other details see legend to Figure 1. MtDNA haplogroups in Iran Over 90% of the mtDNAs found in Iran belong to haplogroups HV, TJ, U, N1, N2 and X, commonly found in West Eurasia (Table 2 ). In contrast to Europe, where H is predominant among the mtDNA haplogroups, in Iran the frequency of haplogroup U (29%) is higher than that of haplogroup H (17%) (Table 9, see Additional file 4 ). This difference accounts, at least partly for the presence in Iran of U sub-groups, such as U7 (9.4%), that are virtually absent in Europe. Compared to India, haplogroup M frequency in Iran is marginally low (5.3%) and there are no distinguished Iranian-specific sub-clades of haplogroup M. All Iranian haplogroup M lineages can be seen as derived from other regional variants of the haplogroup: eleven show affiliation to haplogroup M lineages found in India, twelve in East and Central Asia (D, G, and M8) and one in northeast Africa (M1). Indian-specific (R5 and Indian-specific M and U2 variants) and East Asian-specific (A, B and East Asian-specific M subgroups) mtDNAs, both, make up less than 4% of the Iranian mtDNA pool. We used Turkey (88.8 ± 4.0%) as the third parental population for evaluating the relative proportions of admixture from India (2.2 ± 1.7%) and China (9.1 ± 4.1%) into Iran. Therefore we can conclude that historic gene flow from India to Iran has been very limited. The package of the most ancient mtDNA haplogroups in India Approximately one tenth of the Indian haplogroup M mtDNAs fall into its major sub-clade M2, which is defined by the motif 477G-1780-8502-16319 [ 15 ]. M2 can be further subdivided into haplogroups M2a (transitions at nps 5252 and 8369) and M2b [ 15 ]. Haplogroup M2 and its two major sub-clades reveal coalescence times of 50 to 70 thousand years (Table 3 ). Due to the increased frequency towards the southern part of India (Figure 1 , panel M2, SAA p < 0.05 Figure 4 ), M2 is significantly (p < 0.05) more frequent among the Dravidic speakers than among the Indo-European speakers who are spread mostly in the northern regions of India (Table 2 ). It is more plausible that geography rather than linguistics is behind this pattern, because the frequency of M2 amongst the Indo-European speaking populations in southern India is significantly higher than that in the north, while there is no significant difference between Dravidic and Indo-European speaking populations from the same geographic region (Table 2 ). It is also notable that the frequency of M2 among the Brahmins and the Kshatriyas of Andhra Pradesh (CR 3.3 – 19.2%) is not significantly (p > 0.05) different from that among the other castes or the tribal populations of the region (CR: 5–12.9%, 11.2–18.3%, respectively). On the other hand, none of the 159 Brahmins and Kshatriyas from the northern states of India (Punjab, Rajasthan, Uttar Pradesh and West Bengal) belong to M2 while the frequency reaches nearly 3% (CR: 1.6–4.6%) among the other castes and tribal populations of the region. Table 3 Indian-specific sub-clades of mtDNA haplogroups M and R. DIAGNOSTIC CODING REGION MARKERS ANCESTRAL HVS-I MOTIF COALESCENCE (years) ρ/nδ 2 a COALESCENCE b (years) ρ/nδ 2 a n c Proportion (95% CR) M2 477G-1780-8502 16223–16319 70,600 ± 21,000 0.02 70,100 ± 20,700 0.04 166 6.1 (5.3–7.1) M2a 5252–8369 16223-16319-16270 48,300 ± 20,100 0.03 46,700 ± 22,800 0.06 79 2.9 (2.3–3.6) M2b 16223-16319-16274 54,800 ± 25,000 0.02 57,600 ± 22,300 0.06 87 3.2 (2.6–3.9) M3a 4580 16126–16223 17,300 ± 7,400 0.10 17,300 ± 7,600 0.11 62 3.0 f (2.4 – 3.8) M4a 6620–7859 16223–16311 19,200 ± 9,000 0.23 19,100 ± 9,000 0.25 21 0.8 (0.5–1.2) M6 3537 16223-16231-16362 33,000 ± 13,900 0.04 30,000 ± 13,600 0.08 79 2.9 (2.3–3.6) M6a 16223-16231-16356-16362 19,100 ± 7,600 0.17 15,700 ± 8,100 d 0.54 37 1.4 (1.0–1.9) M6b 5585 16188-16223-16231-16362 6,000 ± 2,100 0.73 12,100 ± 4,500 0,82 37 1.4 (1.0–1.9) M18 16223-16318T 9,400 ± 3,200 0.31 17,100 ± 4,700 0.61 58 2.1 (1.7–2.7) M25 15928 16223–16304 19,400 ± 7,200 0.30 22,300 ± 8,600 0.32 25 1.0 g (0.7 – 1.5) R2 e 4216 16071 40,400 ± 14,300 0.45 52,500 ± 21,700 0.5 8 0.3 (0.2–0.6) R5 8594 16266–16304 66,100 ± 22,000 0.04 69,800 ± 24,800 0.05 58 2.1 (1.7–2.7) R6 -12282 AluI 16129–16362 30,000 ± 11,000 0.14 30,300 ± 11,600 0.19 35 1.3 (0.9–1.8) Total 2719 a Efficiency of the sample for coalescence calculation (Saillard et al. 2000) b Calculated without the tribal populations c In the Indian subcontinent (a total of 2719 mtDNAs) d Excluding also the scheduled cast of Mukri e R2 is spread also in the Near East, Central Asia and Volga basin f Excluding the datasets where M3* / M3a discrimination was impossible: 246 subjects from Andhra Pradesh (Bamshad et al. 1998); 382 Pushtoon, Koragas, Naga, Yerava, Pardhi, Paniya Adi, Andh, Apatani and Soligas (Cordaux et al. 2003); 35 Kota and Kurumba (Roychoudhury et al. 2001) g Excluding the datasets where M* / M25 discrimination was impossible: 246 subjects from Andhra Pradesh (Bamshad et al. 1998); 39 Thoti and 29 subjects from Bangladesh (Cordaux et al. 2003); 12 Muria (Roychoudhury et al. 2001) Figure 1 The spatial distribution of Indian-specific mtDNA haplogroups (M2, M6 and M3) and their sub-haplogroups. Maps of South and Southwest Asia depicting the spatial frequency distribution of various Indian-specific mtDNA haplogroups. For India, the tribal populations were excluded (see 'Methods' for explanation). The published (full reference in Table 6, see Additional file 1) and new data were averaged to the resolution of states in India, geographic regions in Iran and provinces in China and Thailand. Numbers adjacent to the data points indicate sample sizes. In the case of haplogroup M3a some datasets had to be excluded because the discrimination between M* and the respective subgroup was not possible on the basis of the HVS-I data alone (see footnote of Table 3). We found that R5, which is defined by transitions at nps 8594 [ 27 ], 16266 and 16304, is the second most frequent sub-clade of R in India after haplogroup U. The coalescence age estimate for R5 was similar to that of M2 (Table 3 ), whereas individual boughs within the R5 limb showed expansions from ca. 20,000 ybp to ca. 50,000 ybp (Figure 12, see Additional file 8 ). Our data indicate that this diverse and ancient haplogroup is present over most of India (Figure 3 , panel R5). Though absent among the Austro- Asiatic tribal groups, R5 is spread across the other language boundaries. In contrast to M2, R5 is more frequent among the caste (CR: 2.5–4.5%) than among the tribal populations (CR: 0.7–1.9%) (Table 2 ). Figure 3 The spatial distribution of Indian-specific mtDNA haplogroups (R5 and R6) and West Eurasian-specific U7, W and R2 in South and southwest Asia. For other details see legend to Figure 1. Together with the Indian-specific sub-clades of haplogroup U2 [ 13 , 27 ], haplogroups M2 and R5 can be discriminated as a package of Indian-specific mtDNA clades harboring extremely deep coalescence times (around 50,000 – 70,000 ybp). Together they constitute nearly 15% of the Indian mtDNAs. Importantly, these haplogroups are virtually absent elsewhere in Eurasia [ 13 , 15 ], this study]. Because most of Indian varieties of haplogroup M are still unclassified (M), this package is likely to be extended when more mtDNA coding region information will become available for the M lineages in India. The geographic distribution of the M2, R5 and U2i package seems to be rather uniform in the context of the Indian-specific maternal lineages (SAA p > 0.05). When excluding the mtDNAs that are likely to have arrived more recently from West or East Eurasia, the share of the package among the caste populations in northern and southern India is roughly similar (CR: 16.1–24.5% and 19.5–26.0 %, respectively). However, in accordance with expectations from the individual haplogroup distributions, the tribal groups speaking Austro-Asiatic and Tibeto-Burman languages are characterized by considerably lower values (CR: 3.9–14.1% and 3.8–15.1%, respectively). The quest for finding the origin of haplogroup M and a plausible scenario for the peopling of Eurasia Based on the high frequency and diversity of haplogroup M in India and elsewhere in Asia, some authors have suggested ( versus [ 3 ]) that M may have arisen in Southwest Asia [ 16 , 17 , 31 ]. Finding M1 or a lineage ancestral to M1 in India, could help to explain the presence of M1 in Africa as a result of a back migration from India. Yet, to date this has not been achieved [ 15 ], this study). Therefore, one cannot rule out the still most parsimonious scenario that haplogroup M arose in East Africa [ 3 ]. Furthermore, the lack of L3 lineages other than M and N (indeed, L3M and L3N) in India is more consistent with the African launch of haplogroup M. On the other hand, one also observes that: i) M1 is the only variant of haplogroup M found in Africa; ii) M1 has a fairly restricted phylogeography in Africa, barely penetrating into sub-Saharan populations, being found predominantly in association with the Afro-Asiatic linguistic phylum – a finding that appears to be inconsistent with the distribution of sub-clades of haplogroups L3 and L2 that have similar time depths. That, plus the presence of M1 without accompanying L lineages in the Caucasus [ 32 ] and [our unpublished data], leaves the question about the origin of haplogroup M still open. In contrast to haplogroup M, ancient sub-clades of haplogroup N are spread both east and west of India as well as within India itself. Several migration scenarios involving multiple "out of Africa" events punctuated by space, time or both, could be invoked to explain the phylogeography of these mtDNA haplogroups. Yet, using the parsimony criterion it can be argued that only a single early migration that brought ancestral lineages, M and N (with the latter having already given rise to R), to South Asia could account for the extant mtDNA phylogeography in Eurasia [ 15 ]. The finding of several largely South and West Asian-specific sub-clades (H, L, R2, and F) of the major Eurasian Y chromosomal haplogroups F and K also supports this scenario [ 15 ]. From South and West Asia the colonization would have sprung both east and west as region-specific mtDNA and Y chromosomal sub-clades appeared both in West- and East-Eurasia as well as in India itself (Figure 5 ). However, not all the West-Eurasian Y-chromosomal founder haplogroups are present in India. Haplogroup E, for example, was possibly carried to Europe and Western Asia later via the Levantine corridor [ 33 ]. Similarly, a Late Upper Palaeolithic origin and spread of mtDNA haplogroup X from Northeast Africa and Middle East has been suggested lately [ 34 ]. Figure 5 Peopling of Eurasia. Map of Eurasia and northeastern Africa depicting the peopling of Eurasia as inferred from the extant mtDNA phylogeny. The bold black arrow indicates the possible "coastal" route of colonization of Eurasia by anatomically modern humans (ca. 60,000 – 80,000 ybp.). This "Southern Coastal Route" is suggested by the phylogeography of mtDNA haplogroup M, the virtual absence of which in the Near East and Southwest Asia undermines the likelihood of the initial colonization of Eurasia taking a route north around the Red Sea. Therefore, the initial split between West and East Eurasian mtDNAs is postulated between the Indus Valley and Southwest Asia. Spheres depict expansion zones where, after the initial (coastal) peopling of the continent, local branches of the mtDNA tree (haplogroups given in the spheres) arose (ca. 40,000 – 60,000 ybp), and from where they where further carried into the interior of the continent (thinner black arrows). Admixture between the expansion zones has been surprisingly limited ever since. We note that while there is no obvious need to introduce the "northern route" – from northeast Africa over Sinai to the Near East – to explain the initial colonization of Eurasia, the spread of some mtDNA and Y-chromosomal haplogroups implies that the "northern" passage might have been used in a later period [33, 34]. The improved structure of autochthonous Indian mtDNA clades Nearly a third of Indian mtDNAs belonging to haplogroup M could be assigned to its existing boughs and limbs with the current knowledge of the mtDNA coding region polymorphisms (Table 9, see Additional file 4 ). It is likely that the unclassified Indian M and R* mtDNAs are also to a large extent autochthonous because neither the East nor West Eurasian mtDNA pools include such lineages at notable frequencies. Haplogroup M6 (Figure 6 ) is primarily found in the Indus Valley and on the western shores of the Bay of Bengal where its sub-clades M6a and M6b are concentrated towards the southwest and the northeast, respectively (Figure 1 , panel M6, M6b cline is significant SAA p < 0.05, Figure 4 ). The highest frequencies of M6a and M6b were found amongst the Mukri scheduled caste from Karnataka (17%) and in Kashmir (10%), respectively. The Mukri form an endogamous group of no more than 10,000 individuals, who dwell on an area less than 2000 km 2 [ 35 ]. That, together with the observation that all the sixteen M6 sequences found among the Mukri belong to a single haplotype, suggests that genetic drift has played a major role in the demographic history of the Mukri. The statistical significance of the high M6 frequency in Kashmir is undermined by the small sample size (19 individuals), which results in the very wide error margins for the frequency estimate (CR: 3.2–31.7%). Figure 6 Network of HVS-I haplotypes belonging to haplogroup M6. Circle areas are proportional to haplotype frequencies. Variant bases of the HVS-I are numbered as in (Anderson et al. 1981) minus 16,000 and shown along links between haplotypes. Character changes are specified only for transversions. Underlined samples are those in which the diagnostic coding region markers (3537 and 5585) were assayed by either RFLP analysis (M6: -3537 AluI; M6b: -5584 AluI) or direct sequencing. Sample codes are as in Table 6 (see Additional file 1). Coalescence estimates marked with an asterisk are calculated excluding tribal populations (see Materials and Methods for explanation). The coalescence estimate marked with two asterisks is calculated without the data on tribal and scheduled caste (the Mukri) populations (see text for details). Different geographic distributions characterize the sub-clades of M3 and M4 that we define by mtDNA coding region markers (Table 3 , Figures 7 and 8 ). Both M3a and M4a show time depths around 20,000 ybp. However, while M4a is sparsely spread in most of India with no obvious geographical cline (Figure 2 , panel M4a, SAA p > 0.05), the spread of M3a is concentrated into northwestern India (Figure 1 , panel M3a, SAA p < 0.05), suggesting that the region may have been the ancestral source. The frequency of M3a is at its highest amongst the Parsees of Mumbai (22%). Given the low M3a diversity amongst the Parsees – the twelve M3a mtDNAs fall into the two most common haplotypes (Figure 7 ) – the high frequency is likely a result of admixture and subsequent founder events. On the other hand, it is intriguing that, despite its low frequency, M3a penetrates into central and southwestern Iran (Figure 1 , panel M3a) – the historic origin of the Zoroastrian Parsees. In addition to the Parsees we found M3a at high frequencies amongst the Brahmins of Uttar Pradesh (16%) and the Rajputs of Rajasthan (14%) (Table 10, see Additional file 5 ). Figure 7 A network of haplogroup M3a haplotypes. Underlined samples are those in which the diagnostic coding region marker (4580) of M3a was assayed by either RFLP analysis (-4577 NlaIII) or direct sequencing. For other details, see the legend to Figure 6. Figure 8 Network of HVS-I haplotypes belonging to haplogroup M4a. Underlined samples are those in which the diagnostic coding region marker (6620) of M4a was assayed by either RFLP analysis (+6618 MboI) or direct sequencing. For other details, see the legend to Figure 6. Awaiting further information from complete mtDNA sequences, we defined haplogroup M18 by using the transversion at np 16318. This star-like cluster (Figure 9 ) is spread at low frequencies across India, with the exceptions of the very north and the coast of the Arabian Sea (Figure 2 , panel M18). The high incidence (33%) of the M18 nodal haplotype among the Austro-Asiatic speaking Lodha of West Bengal (Table 10, see Additional file 5 ) suggests a possible founder effect in this population. This explains the nearly two-fold difference between the coalescence estimates for this cluster calculated with and without the tribal data (Table 3 ). Figure 9 Network of HVS-I haplotypes belonging to haplogroup M18. For other details, see the legend to Figure 6 The G to A transition at np 15928 has been spotted on different branches (e. g. haplogroups T and M) of the mtDNA phylogeny [ 3 , 36 ]. Quintana-Murci and colleagues observed this transition within haplogroup M in combination with the HVS-I motif 16048-16129-16223-16390 [ 27 ]. None of the mtDNAs in our study which harbor – or stem from – the 16048-16129-16223 motif were positive for the 15928 transition, suggesting an additional occurrence. In addition, we recorded this transition associated with three other HVS-I motifs on the background of haplogroup M (M8-Z: 16185-16223-16260-16298; M: 16223 and M: 16086-16223-16335). These occurrences cannot be monophyletic for obvious reasons. Yet, when combined with the transition at np 16304, G15928A roots a star-like subclade of haplogroup M that we tentatively named M25 (Figure 10 ). In this case, monophylecity is the most parsimonious assumption. This haplogroup is moderately frequent in Kerala and Maharashtra but rather infrequent elsewhere in India (Figure 2 , panel M25). Figure 10 Network of HVS-I haplotypes belonging to haplogroup M25. Underlined samples are those in which the diagnostic coding region marker (15928) of M25 was assayed by either RFLP analysis (-15925 MspI) or direct sequencing. For other details, see the legend to Figure 6. Coalescence estimates for these Indian-specific mtDNA haplogroups (M3a, M4a, M6, M25 and R6) fall largely between 20,000 and 30,000 ybp. These estimates overlap with those of many West Eurasian-specific (e.g. H, HV, preHV, U3, U4, K, X [ 9 , 34 ]) and East Eurasian-specific (A, F2, D4, M7c1, M7a1, M8a [ 7 , 22 ]) mtDNA clades, suggesting a rather synchronic worldwide demographic expansion event in the late Pleistocene, during an interglacial period preceding the LGM. Several Indian-specific mtDNA clades demonstrate a similar spread-pattern in southern India. We found haplogroups M4a, M6a and M18 in southeastern Tamil Nadu and Andhra Pradesh while they were absent from neighboring Karnataka and Kerala (Figure 1 panel M6a and Figure 2 panels M4a and M18). One possible explanation is that admixture has been facilitated along the coastlines of the Arabian Sea and the Bay of Bengal. On the other hand, because the absolute frequencies of these haplogroups are rather low, it cannot be ruled out that an increase of sample sizes would disrupt the observed spread-pattern. Were the Austro-Asiatic speaking tribal people the earliest inhabitants of India? By calculating nucleotide diversities and expansion times (using the method from [ 37 ]) for different linguistic groups of India, some previous studies on mtDNA variation have distinguished the Austro-Asiatic speaking tribal groups as the carriers of the genetic legacy of the earliest settlers of the subcontinent [ 17 , 38 ]. However, because the linguistic groups of India do not cluster into distinct branches of the Indian mtDNA tree [ 13 , 15 , 19 ], this study], calculating the beginning of expansion for those groupings is problematic and likely controversial as well. Recently, Basu et al. (2003) supported the conclusions of [ 17 , 38 ] by reporting that the frequency of the ancient haplogroup M2 among the Austro-Asiatic tribal populations is as high as 19%, and that they lack the slightly younger haplogroup M4 (the likely paraphyletic mother-clade of M4a). The authors have regarded the HVS-I transition at np 16319 as sufficient in defining haplogroup M2. This assumption, however, might lead to an overestimation of M2 frequency and age. Indeed, the 16319 transition has arisen several times on the background of other Indian haplogroup M lineages (Table 7, see Additional file 2 ), more specifically, in mtDNAs lacking the coding region markers that define M2 [ 15 ]. Although two out of the four M2 sequences reported by Basu et al. (2003) among the Lodha, Mundas and Santals (HVS-I sequences originally published by [ 17 ]) do harbor the characteristic M2a HVS-I motif (16223-16270-16274-16319-16352), without information from the coding region it is not clear whether the other two sequences (HVS-I motifs: 16092-16179-16223-16289-16294-16319 and 16147G-16172-16223-16319) represent novel M2 sub-clades (because these sequences cannot be affiliated with M2a or M2b) or derive from two independent branches of haplogroup M where 16319 transition has arisen recurrently. HVS-I motif 16147G-16172-16223, for example, is commonly associated with haplogroup N1a. Since sequence data on the five M2s among the Austro-Asiatic speaking tribe Ho, reported by Basu et al. (2003), have not been made available in the publication, we cannot rely on their haplogroup classification. Thus, we are left with one Munda and one Santal mtDNA belonging to haplogroup M2. They make up just 5% of the Austro-Asiatic tribal sample of 37 subjects (excluding the ten Ho). Interestingly, we found no instances of haplogroup M2 among the 56 Lodhas analyzed in this study. Consequently, when excluding the recurrences of the 16319 transition on the background of other sequence motifs, the frequency of M2 among the Austro-Asiatic speaking tribal groups from West Bengal in the combined dataset (Table 7, see Additional file 2 ) is significantly reduced to about 2%. The corrected value is comparable to the M2 frequency (>3%) in tribal populations speaking Indo-European languages of Punjab and Uttar Pradesh, but is significantly lower than its frequency (>14%) among the Dravidic-speaking tribal groups of Andhra Pradesh (Table 8, see Additional file 3 ). Language families present today in India, such as Indo-European, Dravidic and Austro-Asiatic, are all much younger than the majority of indigenous mtDNA lineages found among their present-day speakers at high frequencies (see Additional file 9 ). It would make it highly speculative to infer, from the extant mtDNA pools of their speakers, whether one of the listed above linguistically defined group in India should be considered more "autochthonous" than any other in respect of its presence in the subcontinent. Additionally, we note that some recent linguistic and archaeological evidence place the spread of the Austro-Asiatic languages in the Neolithic, in conjunction with the dispersal of rice cultivation from the Yangtze River basin [ 39 ]. If this were the case, it would imply that the arrival of this linguistic phylum in India was not associated with female gene flow. Gene flow from West Eurasia Broadly, the average proportion of mtDNAs from West Eurasia among Indian caste populations is 17% (Table 2 ). In the western States of India and in Pakistan their share is greater, reaching over 30% in Kashmir and Gujarat, nearly 40% in Indian Punjab, and peaking, expectedly, at approximately 50% in Pakistan (Table 11, see Additional file 6 , Figure 11 , panel A). These frequencies demonstrate a general decline (SAA p < 0.05 Figure 4 ) towards the south (23%, 11% and 15% in Maharashtra, Kerala and Sri Lanka, respectively) and even more so towards the east of India (13% in Uttar Pradesh and around 7% in West Bengal and Bangladesh). The low (<3%) frequency of the western Eurasian mtDNAs in Rajasthan may be in part a statistical artifact due to the limited sample size of 35 Rajputs. Figure 11 The segregation of West Eurasian, East Eurasian and South Asian mtDNA pools. Partial map of Eurasia illustrating the spatial frequency distribution of mtDNA haplogroups native to West Eurasia (panel A), South Asia (panel B) and East Eurasia (panel C). Data points represent states in India, geographic divisions in Iran and provinces in China and Thailand. Numbers adjacent to the data points indicate the applicable sample size. The distribution of West Eurasian-, South Asian- and East Eurasian-specific mtDNA haplogroups amongst the tribal populations of South and Southeast Asia are depicted as pie diagrams on panel D (colors as on panels A, B, and C, while the white color represents unclassified M* and R* mtDNAs). The data on tribal populations was not used for the isofrequency maps of panels A, B and C (see Materials and Methods for explanation). In comparison to an overall frequency of 17% (CR 15.1–19.3%) among the caste populations, only 7% (CR 6.2 – 9.0%) of the mtDNAs from the tribal groups show affiliation to the West Eurasian haplogroups. The observed difference could be caused by differences in the extent of gene flow from the west to different social layers of the Indian society [ 19 ], and/or a more pronounced genetic drift among the tribal groups. This western Eurasian contribution into the Indian maternal gene pool can be broadly divided into two different components. Over two thirds of the West Eurasian-specific mtDNAs found in India are made up by haplogroups HV, TJ, N1 and West Eurasian-specific branches of haplogroup U. It is likely that these mtDNA haplogroups have been carried to western India both by relatively low-intensity long-lasting admixture at the border regions as well as a consequence of numerous but probably limited migrations during the last 10, 000 ybp [ 13 ]. The remaining one third of the West Eurasian-specific mtDNAs found in India is comprised of haplogroups U7, R2 and W showing much deeper time depths in India – approximately forty thousand years before present (Table 4 ). A large-scale immigration – carrying these haplogroups – could have introduced a substantial fraction of the diversity already present within the putative source areas. This would explain the deep coalescence times of these haplogroups in India, while their actual arrival could have occurred later. Alternatively, the coalescence estimates may indeed reflect a deeper autochthonous history of these mtDNA clades in India. It is worthwhile to stress that while in India the share of U7, R2 and W in the West Eurasian-specific mtDNAs mounts to nearly a third, in Iran it stays below 15%. Table 4 Coalescence estimates and diversity values for mtDNA haplogroups U7, W and R2 in India, Central Asia, Near and Middle East. INDIA NEAR AND MIDDLE EAST CENTRAL ASIA HAPLO-GROUP COALESCENCE (years) DIVERSITY COALESCENCE (years) DIVERSITY COALESCENCE (years) DIVERSITY U7 41,400 ± 15,800 0.941 41,200 ± 14,800 0.91 34,400 ± 15,500 0.941 W 37,900 ± 11,100 0.883 32,000 ± 8,700 0.934 27,400 ± 8,300 0.758 R2 40,400 ± 14,300 0.923 36,100 ± 11,100 0.955 25,200 ± 13,300 0.889 Similarly to HV, TJ, N1, the spread of U7, W and R2 in India is geographically uneven – the three haplogroups are much more frequent in the northwestern states (SAA p < 0.05, Figure 4 ). Together, they constitute nearly 14% of the maternal gene pool in Indian Punjab, Gujarat and Rajasthan. Their frequency is also high in Iran (13%) and Pakistan (10%) but declines in Central Asia (5%). Although haplogroup W is not highly frequent in European populations, it is nevertheless quite common [ 9 ], reaching the highest frequencies among the central-northern Finns (9% [ 40 ]). Yet, it is virtually absent from the Finno-Ugric speaking populations of the Volga basin [ 41 ]. In Central Asia the frequency of haplogroup W stays below 2%. Many European populations are lacking haplogroup U7 [ 9 ], but its frequency climbs over 4% in the Near East and up to 5% in Pakistan, reaching nearly 10% level in Iranians (Table 9, see Additional file 4 ). In India, haplogroup U7 frequency peaks at over 12% in Gujarat, in the westernmost state of India, while for the whole of India its frequency stays around 2%. Expansion times and haplotype diversities for the Indian and Near and Middle Eastern U7 mtDNAs are strikingly similar (Table 4 ). The possible homeland of this haplogroup spans likely in Gujarat and Iran because from there its frequency declines steeply both to the east and to the west. If the origin were in Iran rather than in India, then its equally high frequency as well as diversity in Gujarat favors a scenario whereby U7 has been introduced to the coastal western India either very early, or by multiple founders. Notably, the overlap of the Indian and Iranian lineages is largely restricted to the ancestral nodes while the coalescence age estimates for the nearly exclusively Indian (16207-16309-16318T) and West Eurasian (16126-16309-16318T) founder HVS-I motifs both yield time depths of about 20,000 to 30,000 ybp. Haplogroup R2 appears at low frequencies in Near and Middle East and India and is virtually absent elsewhere. The spread of haplogroup R2 in Europe is restricted to a few populations in the Volga basin where it is represented by nodal haplotypes and by a region-specific subclade characterized by the HVS-I motif 16037–16172 [ 41 ]. The coalescence estimate of this sub-clade is 11,400 ± 9,000 ybp. However, its wide error range prevents us from drawing any firm conclusions. To summarize, the West and South Asian phylogeography of haplogroups W, U7 and R2 can be viewed as a genetic continuum that spans from the Near East into India, extending north into Central Asia. The coalescence times of these haplogroups suggest that this continuum took shape somewhere between 30,000 to 50,000 ybp (Table 4 ), thus falling within the climatically favorable interglacial period. We notice that the extant U7 and W frequencies along the proposed continuum are not uniform. U7 is more predominant in Iran, Pakistan, northwestern India and the Arabian peninsula, while W is more frequent in the western Near-East, Anatolia and the Caucasus. The coalescence ages of the Indian- and Iranian-specific U7 clades suggest that the time-window of this continuum was closed by ca. 20,000 ybp. The inferred extreme aridity of eastern Iran and western India during the last glacial maximum, which is well documented in paleovegetation reconstructions [ 42 ] may explain the observed segregation. It has been suggested that the Jews settled in southwest India on the coast of the Arabian Sea sometime during the early Middle Ages. However, the mtDNA pool of the extant Cochin Jews is overwhelmingly Indian-specific (Table 10, see Additional file 5 ). We found exact or close matches to the fourteen HVS-I haplotypes observed among the Cochin Jews in other Indian populations. It is not clear whether the Near Eastern mtDNA lineages have been lost or the initial Jewish settlers did not include women. Gene flow from East Eurasia The East Eurasian-specific mtDNA haplogroups are less common in India and more sharply geographically segregated than the haplogroups of western Eurasian ancestry (Table 2 ; Figure 11 , panel C). Indian caste populations harbor only about 4% of such mtDNAs, compared to 17% of the West Eurasian ones (Table 2 ). Elevated frequencies of haplogroups common in eastern Eurasia are observed in Bangladesh (17%) and Indian Kashmir (21%) and may be explained by admixture with the adjacent populations of Tibet and Myanmar (and possibly further east: from China and perhaps Thailand). On the other hand, the high frequencies of East Eurasian-specific mtDNAs found in the southern Indian state of Tamil Nadu (21%) are unexpected when considering their relatively low frequencies (~1%) in West Bengal and Andhra Pradesh. We notice, however, that the haplogroup assignments used here for the Tamil Nadu sample (A4, B4, F1a and M7) (Table 7, see Additional file 2 ) are based on HVS-I sequences alone [ 29 ]. As shown and discussed elsewhere [ 7 ], such type of assignment is prone to mistakes. Two varieties of haplogroup M, D4c and G2a, were recently identified as largely specific to Central Asia [ 43 ]. In spite of geographical proximity we did not find these haplogroups in northern or northwestern India. Haplogroup G2a did, though in marginally low frequency, turn up in Iran (CR: 0.1 – 1.6%) and in southern Indian states Andhra Pradesh (CR: 0.3 – 2.9%) and Sri Lanka (CR: 0.2 – 4.1%). Tibeto-Burman speaking tribal populations of eastern and northern India exhibit the highest frequencies of East Eurasian-specific mtDNA haplogroups. As inferred from the published HVS-I sequences [ 29 ], their share sums up to approximately two thirds of mtDNAs among the tribal groups in Assam, Nagaland, Arunachal Pradesh and Tripura, (Table 8, see Additional file 3 ; Figure 11 , panel D). MtDNA haplogroups native to East Eurasia are also highly frequent in the northern states of India, reaching a peak of nearly 50% among the Kanet of Himachal Pradesh. Papiha and colleagues have previously demonstrated through the typing of immunoglobin allotypes that the Tibetan admixture among the regional Kanet groups decreases as the distance from the Tibetan border increases [ 44 ]. Thus, mtDNA data are consistent with an ancestral origin of the Tibeto-Burman speaking tribal populations outside (east of) India in the neighboring Tibet and Myanmar [ 30 , 45 ]. Haplotype sharing between populations The majority (70%) of the 1136 mtDNA haplotypes found among continental Indians (including Pakistan and Bangladesh) are singletons and 41% of those that occur more than once are restricted to a single population. Only a few haplotypes are shared among five or more populations. The number of shared haplotypes between pairs of social, linguistic and geographic groups of Indian populations is slightly (but in most cases insignificantly) lower than that between random groups of Indian populations taken for reference (see Materials and Methods). Where the decline of shared haplotypes is significant relative to the reference, it is most probably caused by large differences in the sample sizes of the groups under comparison (Table 5 ). Table 5 Proportion of mtDNA haplotype sharing between population groups of South Asia Group 1 Group 2 pop. group n n b 1/2 d pop. group n n b 2/1 e n c Random 1 g 1256 ± 215 617 ± 109 .399 – .453 Random 2 g 1426 ± 215 643 ± 114 .396 – .447 132 (112 – 154) Tribals 1197 437 .411 – .467 Castes 1485 806 .324 – .372 107 (90 – 128) Northern states 1204 647 .277 – .329 Southern states 1478 585 .345 – .394 96 (80 – 116) Western states 1142 508 .453 – .511 Eastern states 1540 735 .331 – .379 107 (90 – 128) Dravidians 974 380 .428 – 490 Others 1708 846 .273 – .317 90 (74 – 109) Indo-Europeans 1322 686 .328 – .379 Others 1360 556 .393 – .446 106 (89 – 127) AA tribals 90 35 .552 – .746 Others 2592 1110 .014 – .025 9 (5 – 17) TB tribals 249 142 .174 – .277 Others 2433 1018 .091 – .115 24 (16 – 35) Indo-Europeans 1322 686 .295 – .346 Dravidians 974 380 .425 – .487 87 (71 – 106) Total 2682 1136 340 f a continental Indians including Pakistan and Bangladesh (excluding Andaman & Nicobar) b number of haplotypes c number of shared haplotypes between group 1 and group 2 (95% credible regions; CR) d CR of the proportion of those haplotypes in group 1 that occur also in group 2 e CR of the proportion of those haplotypes in group 2 that occur also in group 1 f 41% of the shared haplotypes are shared only between members of the same population g ± indicate SD An alternative method that assesses the degree of haplotype sharing between populations is to investigate the combined frequency of the shared haplotypes in two population groups. Thus, amongst the northern and the southern population groups the combined frequency of the haplotypes present also in the other group is significantly lower than that which we observed in the case of random groups. This is not surprising because West Eurasian-specific mtDNA haplogroups are rather frequent in northwest India. Because the Indo-European and the Dravidic speakers of India are largely concentrated to the northern and southern parts of the subcontinent, respectively, the differences arising from geographic division of the Indian populations also correspond to these linguistic groupings (Table 5 ). Conclusions Three Indian-specific haplogroups, M2, U2i and R5, which encompass about 15% of the Indian mtDNA pool, exhibit equally deep coalescence ages of about 50,000 – 70,000 years. Thus, their spread can be associated with the initial peopling of South Asia. Haplogroups U7, W and R2 harbor a number of similar traits. Their overlapping geographic distributions and coalescence times suggest some degree of genetic continuum in the area spanning from the Near and Middle East through northwest India and reaching north into Central Asia. The coalescence estimates for these haplogroups are equally deep (around 30,000 – 50,000 years) in these different regions. That may be a result of either relatively more recent albeit large in scale migrations that brought along most of the diversity or may indeed reflect the region-specific expansions of these haplogroups. The former explanation could be ruled out since it is impossible to envisage a substantial movement of mtDNAs from South Asia that would not include haplogroup M. The same is true for the opposite – the share of U7, W and R2 within the West Eurasian-specific mtDNA haplogroups is two-fold higher in India than it is in Iran. Moreover, the South- and West Asian-specific sub-branches of haplogroup U7 predate the last glacial maximum. Therefore, deep autochthonous history of these haplogroups in the region remains to be the most parsimonious explanation. Through the use of mtDNA coding region markers, we were able to classify altogether a quarter of the Indian M and R mtDNAs into a number of Indian-specific mtDNA haplogroups, four of which we newly identified. Several of these are characterized by clear patterns in their geographic distribution and/or different frequencies among different socio-cultural groups of India. Additional efforts should be undertaken to identify new coding region markers in order to further improve the classification of Indian mtDNAs. Indeed, much of the information is still hidden by the poor resolution of the Indian mtDNA tree and further piling of HVS-I datasets would add little to deepen our understanding of the demographic history of South and Southwest Asia. We found that haplogroup M frequency drops abruptly from about 60% in India to about 5% in Iran, marking the western border of the haplogroup M distribution. A similarly sharp border cuts the distribution of Indian-specific mtDNA haplogroups to the east and to the north of the subcontinent. We therefore propose that the initial mtDNA pool established upon the peopling of South Asia has not been replaced but has rather been reshaped in situ by major demographic episodes in the past and garnished by relatively minor events of gene flow both from the West and the East during more recent chapters of the demographic history in the region. Methods Subjects MtDNA sequence variation in a total of 796 Indian samples most of which are held in a collection at Newcastle University [ 46 ] was analyzed. The samples cover a wide geographical range that spans from Himachal Pradesh in the north, Sri Lanka in the south, West Bengal in the east and Gujarat in the west (Table 1 ). Tribal populations constitute 15% of the total sample size. The Lodha (n = 56) live mostly in the western part of Midnapore district of West Bengal where they are also known as Kheria or Kharia. Their total population size was ~59000 according to the 1981 census. Their language belongs to the Mundari branch of the Austro-Asiatic language family [ 47 ]. The Kanet (n = 37) make up two thirds of the ~50000 inhabitants of the Kinnaur district in Himachal Pradesh [ 48 ]. Their language belongs to the Himalayan group of the Tibeto-Burman language family [ 47 ]. Five Bhoksa and twenty-six Tharu individuals were included in the present study in addition to those from the same populations that we have previously reported [ 13 ]. Most of the Tharu live in the Terai areas (a belt of marshy land at the foot of the Himalayas) of Nepal (n = 720000). Approximately 96000 reside in Uttar Pradesh and Uttaranchal, Indian states adjacent to Nepal. Around 32000 Bhoksas live in the lowland areas of Uttaranchal and the neighboring Bijnor district of Uttar Pradesh. Both these tribal groups speak languages belonging to the Indo-European phylum [ 47 ]. The non-tribal Indian samples analyzed contained 105 West Bengalis of different caste rank, 58 Konkanastha Brahmins from Bombay, 53 Gujaratis, 50 Moors and 82 Sinhalese from Sri Lanka, 109 Punjabis of different caste rank from the Punjab, 25 Brahmins from Uttar Pradesh, 35 Rajputs from Rajasthan, 55 Parsees from Maharashtra and 100 subjects from Cochin, Kerala (including 45 Jews who have moved to Israel) (Table 1 ). The Iranian sample of 436 individuals was collected in different locations mainly from southwestern and northwestern Iran (Table 1 ). The new Indian mtDNA sequence data was combined with that previously published on Indian populations [ 3 , 13 , 15 , 17 , 18 , 29 , 35 , 49 - 51 ] to produce a pooled dataset (n = 2572) in which the tribal populations constitute slightly over 50% (Table 6, see Additional file 1 ). By including also the data on Iranian and published data on Pakistani [ 13 , 27 , 29 ], Bangladeshi [ 29 ], Chinese [ 7 , 22 , 52 - 54 ] and Thai [ 53 , 55 , 56 ] populations (n = 145, 29, 919 and 552, respectively), a dataset of over 4600 samples spanning from West to East Asia (Table 6 and 7, see Additional files 1 and 2 ) was obtained. In many cases the published data was reanalyzed. Analogy with the newly obtained HVS-I motifs, that were classified into haplogroups using diagnostic markers of the mtDNA coding region, served as basis for haplogroup assignments of the published HVS-I sequences. An even wider range of data was used for the production of the isofrequency maps: the published data from Armenia (n = 192) [ 32 ] and Kyrgyz (n = 95) [ 57 ], and the unpublished data from Yemen (n = 118), Oman (n = 79), Saudi Arabia (n = 205), Ethiopia (n = 270), Uzbekistan (n = 114) and Tajikistan (n = 41). Further details about the mtDNA variation of these populations will be published elsewhere. MtDNA molecular analyses DNA was extracted using standard phenol-chloroform methods [ 58 ]. The hypervariable segment (HVS)-I (between nucleotide positions (np) 16024–16400) of the control region was sequenced in all the 796 Indian and 436 Iranian samples. Preparation of sequencing templates was carried out following Kaessmann et al. [ 59 ]. Purified products were sequenced with the DYEnamic™ ET terminator cycle sequencing kit (Amersham Pharmacia Biotech) and analyzed on ABI 377 DNA or Megabace2000 Sequencers. Sequences were aligned and analyzed with the Wisconsin Package (GCG). In addition, informative mtDNA coding region positions [ 3 , 11 , 27 ] were assayed (Table 7, see Additional file 2 ) in selected individuals from different HVS-I haplotypes to determine haplogroup affiliations. Data analysis Median networks [ 60 , 61 ] were constructed using Network 3.111 and Network 2.10B programs [ 62 ] with default settings (r = 2; ε = 0). MtDNA coding region markers were given five times the weight of the HVS-I positions. Coalescence of mtDNA haplogroups and sub-haplogroups was calculated using ρ (the averaged distance to a specified founder haplotype) and a mutation rate of one transition per 20,180 years between nps 16090–16365 [ 63 ]. Standard errors for coalescence estimates and efficiency of a sample for coalescence time calculation (ρ/nδ 2 ) were calculated following [ 64 ]. Coalescence times were further calculated with the exclusion of tribal populations sequences, yet preserving the cluster topology (coalescence times marked with an asterisk). A more intense genetic drift (particularly founder effects) could introduce a bias into the coalescence time calculation (for example, see below the coalescence time of haplogroup M18 with and without the Lodha sample). The software kindly provided by Vincent Macaulay was adopted in order to calculate the 95% credible regions (CR) from the posterior distribution of the proportion of a haplogroup/sub-haplogroup in the population. Haplotype diversity was estimated as where n is the number of sequences, k the number of distinct haplotypes, and n i number of sequences with one distinct haplotype. Haplogroup isofrequency maps were generated using the Kriging method in Surfer 7 program of Golden Software. Haplogroup frequencies were averaged over populations from the same state in India, provinces in China and Thailand and geographic divisions in Iran. The data points for Kriging are shown as black dots, while the sample size applicable to the data point is given adjacent to the dot. In relatively small and isolated groups (e.g. tribal groups) random genetic drift might seriously affect the haplogroup frequencies, which may become uninformative when a whole region (e.g. state) is considered (e.g. M18 among the Lodha, see below). Therefore, the tribal data were excluded from the haplogroup isofrequency maps calculation. When illustrating the spread of mtDNA haplogroups native to West Eurasia, East Eurasia and India (Figure 11 , panel D) we present these data as pie diagrams. The respective sample size and origin are indicated adjacent to the diagrams. Spatial autocorrelation analysis was done using the PASSAGE software packet [ 65 , 66 ]. The correlograms of Moran's I autocorrelation coefficient were calculated using binary weight matrix with five distance classes. Data on tribal populations was not used (see previous paragraph for explanation). Haplogroup frequencies based admixture proportions were calculated using the ADMIX 2.0 software [ 67 ]. Haplotypes were defined as the HVS-I motif combined with the haplogroup label for haplotype analysis. In this way it was possible to discriminate between mtDNAs with identical HVS-I motifs but otherwise known to belong to different haplogroups (e.g. sequences with the CRS motif in HVS-I belonging to haplogroups H or R). In order to assess the results of haplotype sharing analysis between population groups (e.g. tribal and caste populations) we divided the Indian populations ten times randomly into two sets and analyzed the level of haplotype sharing between these sets. Authors' contributions SM and SSP collected most of the Indian samples (in 1970's) and have been keeping the collection in Newcastle University, United Kingdom. MM, TK, GH and KK carried out the RFLP analysis and mtDNA sequencing of the majority of the Indian samples. EM, JP, PS and MK carried out the RFLP analysis and mtDNA sequencing of the Iranian samples. DMB collected the DNA and analyzed mtDNA variation amongst the Indian samples from Cochin and the Cochin Jews collected (the latter) in Israel under supervision of KS, who also contributed to the editing of the manuscript. AT provided some critical new information and, in addition to KS, MTPG and PE, engaged into valuable discussions during manuscript preparation. MM did most of the phylogenetic analysis and was in charge of manuscript writing while TK and RV contributed significantly during both stages and were responsible for conceiving and designing the study. All authors read and approved the final manuscript. Supplementary Material Additional File 3 Table 8. Excel spreadsheet. MtDNA haplogroup frequencies among tribal populations. Frequencies of mtDNA haplogroups amongst the tribal populations of India, China and Thailand as averaged over states of India and provinces in China and Thailand. Click here for file Additional File 7 Table 12. Excel spreadsheet. Comparison of the mtDNA haplogroup frequencies amongst the Indian caste and tribal populations. Click here for file Additional File 4 Table 9. Excel spreadsheet. MtDNA haplogroup frequencies in India and Iran. Click here for file Additional File 6 Table 11. Excel spreadsheet. Frequencies of mtDNA haplogroups amongst the Indian caste populations as averaged over states of India. Click here for file Additional File 8 Figure 12. Image file in PNG format. Network of HVS-I haplotypes belonging to haplogroup R5. Circle areas are proportional to haplotype frequencies. Variant bases of the HVS-I are numbered as in (Anderson et al. 1981) minus 16,000 and shown along links between haplotypes. The diagnostic R5 coding region marker 8594 is shown in bold and numbered as in (Anderson et al. 1981). Character changes are specified only for transversions. Underlined samples are those in which the marker 8594 was assayed by either RFLP analysis (-8592 MboI) or direct sequencing. Sample codes are as in Table 6 (see Additional file 1). Coalescence estimates marked with an asterisk are calculated excluding tribal populations (see Materials and Methods for explanation). Click here for file Additional File 5 Table 10. Excel spreadsheet. Frequencies of mtDNA haplogroups amongst the Indian populations. Click here for file Additional File 2 Table 7. Excel spreadsheet. MtDNA variation in the studied populations (raw data). MtDNA control and coding region variation in the populations that were used in the study. The database includes both the newly obtained datasets and the previously published datasets. The latter were in many cases reanalyzed both in silico and by typing for additional mtDNA coding region markers. Click here for file Additional File 1 Table 6. Excel spreadsheet. The list of studied populations. List and details of the populations whose mtDNA were used in the study. This includes both newly obtained datasets and previously published datasets Click here for file Additional File 9 Table 13. Excel spreadsheet. Frequencies of mtDNA haplogroups amongst different linguistic groupings of Indian populations. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC516768.xml
550649	Comparative analysis of mouse skeletal muscle fibre type composition and contractile responses to calcium channel blocker	Background In this study, we examined the correlation between excitation-contraction coupling characteristics and skeletal muscle fibre type by (1) localizing the distribution of dihydropyridine receptor (DHPR) protein and (2) comparing the effect of DHPR blocker on muscles with different fibre type composition, in order to better understand the differences between contractile phenotypes of fibres and to explain the contradictory reports to date on the interaction of dihydropyridines with skeletal muscle isoform of DHPR. Results Histochemical experiments revealed that fluorophore conjugated dihydropyridines stain selectively the membranes of muscle fibres. The staining was most evident in type IIA fibres. The major fibre type in gluteus and femoris, revealed by mATPase staining, was IIA (45.0 and 38.1 %, respectively). In gastrocnemius the content of IIA fibres was 22.7 %. Contraction forces before and after the addition of blocker for the three muscles investigated were: gluteus 0.075 ± 0.017 N vs. 0.052 ± 0.011 N, femoris 0.045 ± 0.005 N vs. 0.033 ± 0.005 N and gastrocnemius 0.089 ± 0.016 N vs. 0.075 ± 0.014 N, respectively. The attenuation of contraction force proportional to the cross-sectional area of the muscle was significantly (P = 0.023) higher in gluteus (28.3 ± 3.5 %) and femoris (27.6 ± 3.2 %) as compared to gastrocnemius (16.1 ± 2.5 %). However, no significant change in the control measurements was observed ruling out the possibility of fatigue. Conclusion The results indicate that the attenuation of the contraction force was largest in muscles with a high percentage of type IIA fibres. This supports our finding that the abundance of dihydropyridine receptors of IIA fibres outnumbers that in the other fibre types. The present data show that the correlation of density of dihydropyridine receptors can be one of the important factors influencing the overall contractile properties of the muscle and for its part explain the contradictory results of previous studies on coupling process.	Background One of the features characterizing mammalian skeletal muscle tissue is the structural variability of muscle fibres. The inclusive properties of skeletal muscle lead to functional diversity of muscle fibres, which has been related to differences in relative proportions of membrane structures and to different amounts of contractile proteins [ 1 ]. Although the interfibre-type differences are well recognized, very little is known about the characteristics of the process leading from electrical membrane excitation to contraction (E-C coupling) related to different cell types. The first indication of variation in E-C coupling between representatives of different fibre types came from studies using vaseline-gap technique in order to record slow calcium currents and asymmetric charge movement in fast- and slow-twitch muscles [ 2 ]. The results suggested functional and structural differences between fast- and slow-twitch mammalian muscles with respect to dihydropyridine receptor (DHPR) density. Recently Goodman et al [ 3 ] provided the first evidence about E-C coupling characteristics being related to myosin heavy chain based fibre type and the muscle from which the fibre originated. Our finding about the density of DHPR varying between different fibre types [ 4 ] supports the hypothesis that optimum contractile function of skeletal muscle is related to its fibre type composition via differences in E-C coupling. Voltage-dependent dihydropyridine receptors play an important role in the function of skeletal muscle. As well known, contraction is initiated by a depolarization of the transverse tubular (T-tubular) membrane, which in turn causes calcium to be released from the stores of sarcoplasmic reticulum (SR). The DHPR is located in the T-tubule membrane acting as a voltage sensor. Linked to the ryanodine receptor located in the sarcoplasmic reticulum membrane, this channel protein triggers the intracellular Ca 2+ -release via the ryanodine receptor [ 5 ]. The DHPR itself is a heteromultimer composed of 5 subunits; α 1 (170 kDa), α 2 (140 kDa), β (55 kDa), γ (33 kDa) and δ (24–33 kDa) [ 6 , 7 ]. The pore forming α 1 subunit contains the receptor for dihydropyridines (DHPs) and other calcium antagonists. DHPs, such as nifedipine, nitrendipine, and nimodipine, bind to DHP receptor in a specific, saturable and reversible fashion. In addition, receptors show high affinity for DHP ligands. The dissociation constant ( K D ) for skeletal muscle membranes is usually in the range of 1–10 nM [ 8 ]. Dihydropyridines are used clinically as blockers in the treatment of hypertension and coronary heart disease [ 9 ]. Besides being the voltage sensor for Ca 2+ release needed in E-C coupling, the α 1 -subunit of the molecule functions also as a calcium channel [ 10 ] giving rise to a slow inward calcium current [ 11 ]. However, DHPR appears to be unimportant as an ion-conducting channel [ 12 ]. Firstly, the calcium current is activated too slowly to generate contraction of the fibre. Second, the contraction continues even if the extracellular calcium is removed, and finally, blocking the slow calcium current through the channel does not prevent contraction. On the other hand, Beam et al [ 5 ] reported a notable increase in the magnitude of inward calcium current in developing muscle thus implicating an important role of the current in maturation. In the present study we examined the E-C coupling characteristics of muscles with different dihydropyridine receptor expression defined by fibre type composition. To test the hypothesis that muscles with different cell type compositions, i.e. DHPR quantities, react differently to calcium channel blocker, we measured contraction forces of three muscles before and after adding the blocker solution. We found variation in the activities of muscles depending on the cell type composition of the muscles. The findings confirm the previously observed pattern and elucidate the role of dihydropyridine receptor in muscle cell contraction. Results Myosin heavy chain analysis and fibre typing In order to analyse the distribution of cell types in the gluteus maximus (GLU), rectus femoris (RF) and gastrocnemius (GAS) muscles, tissue samples were collected and subjected to SDS-PAGE. The different muscles of mouse contained four myosin heavy chain (MHC) isoforms in the increasing electrophoretic mobility, MHCIIa, MHCIId, MHCIIb and MHCI (Fig. 1 ) as compared to those of rat gastrocnemius muscle (in this report MHC isoform expressed in fibre is identified by roman numeral and lower case letter, while the fibre type is identified by a roman numeral and a corresponding capital letter according to Hämäläinen and Pette [ 13 ]). The distribution of these myosin heavy chain isoforms differed among the three muscles studied (Table 1 ). In GAS, the major isoform was MHCIId (35.6 %), expressed together with smaller amounts of IIb (28.1 %). On the other hand, the quantity of MHCIIa was smaller (20.6 %). In contrast to GAS, both GLU and RF were characterized by higher contents of MHCIIa (26.6 % and 27.7 %, respectively), together with MHCd (34.3 %; 23.7 %) and lower amount of MHCIIb (15.9 %; 24.3 %). All the muscles contained also portions of MHCI (GAS: 15.7 %, GLU: 23.2 %, RF: 24.3 %). A statistically significant difference (P < 0.05) was found in the portion of IIb between GAS and GLU as well as in IId between GAS and RF. Four fibre types were also identified in muscle sections stained with mATPase (Table 1 ). The major fibre type in GAS was IID (51.5 %). However, in contrast to the results from SDS-PAGE, IIA fibre type (22.7 %) outnumbered the IIB (18.2 %). The most frequent fibre type in both GLU and RF was IIA (45.0 %; 38.1 %), followed by IIB (31.1 %) in GLU and IID (21.6 %) in RF. There was a statistically significant difference (P < 0.05) in the portion of IIA between GLU and RF. In addition, between GAS and RF a significant difference was found in the proportion of I (P < 0.05) and IID (P < 0.01). Between GAS and GLU a significant difference was found in proportion of IIA (P < 0.05) and IID (P < 0.01). To summarise, the significant difference in proportion of IIA between GAS and RF/GLU is particularly relevant to the present study. DHPR expression and correlation to fibre types We have previously shown that the density of dihydropyridine receptors (L-type calcium channels) in T-tubule membranes increases markedly during the postnatal development of mouse skeletal muscle [ 4 ]. Furthermore, the findings showed that the fast oxidative glycolytic (FOG) fibre type has the most evident appearance of DHPRs. Fig. 2A shows localization of DHPR in mouse RF muscle. DHPR is strongly expressed in some muscle fibres whereas weakly expressed at the membrane of others. The corresponding control slide, preincubated with nifedipine resulted in a loss of staining (Fig. 2C ). To correlate the DHPR expression with muscle fibre typing, histochemical experiments were performed as described. According to the staining intensity, IIA fibre type corresponds to the muscle fibres that strongly express DHPRs (Fig. 2B ). Force measurements GLU, RF and GAS muscles were weighed and the length and diameter were measured. Table 2 shows some morphological details for the muscles studied. To reveal the function of L-type calcium channels in muscles with different kinds of cell type distributions, the single twitch forces of the muscles were measured before and after the addition of a specific channel blocker nifedipine. Maximum contraction forces in proportion to the cross-sectional area of the muscle, and the average responses to 1 μM nifedipine solution are shown in Fig. 3 . The force production of GAS (0.089 ± 0.016 N) was considerably higher in comparison to the ones of GLU (0.075 ± 0.017 N) and RF (0.045 ± 0.005 N). The effect of nifedipine on contractile force was inhibiting in all the muscles studied. The in vitro nifedipine effect on the muscle force production was determined as the percentage of the remaining channel mediated contraction force. The weakening of contraction force proportional to the cross-sectional areas of the muscles was significantly (P = 0.023) higher in GLU (28.3 ± 3.5 %) and RF (27.6 ± 3.2 %) in comparison to GAS (16.1 ± 2.5 %). Experiments performed in increasing concentrations of nifedipine resulted in a clear dose-response curve (Fig. 4 ) and revealed a decreasing force output as the blocker concentration increased. Furthermore, the decrease was significantly smaller in GAS (P < 0.05 at nifedipine concentration of 5 μM), in comparison to the decrease in GLU. Discussion The cell type composition of a muscle is one of the features characterizing the function of the muscle. Type I fibres are most prevalent in muscles involved in the maintenance of posture, whereas type II fibres are used for movements which require high power output. Many vertebrate locomotor muscles are composed of a mixture of fibre types. Additionally, one muscle fibre may contain several myosin heavy chain types indicating heterogeneity at fibril level [ 14 ]. The m. gastrocnemius (whole) used in this study has a high type IIB content whereas both m. rectus femoris and m. gluteus maximus are predominantly composed of types IIA and IID. The results from myosin heavy chain analysis and fibre typing are somewhat different. The identification of four different fibre types using histochemical methods is difficult because of the presence of several types of myosin heavy chains within one single fibre. Therefore, the MHC analysis is more qualitative. The correlation of high density of DHPRs with IIA fibres was stated on the basis of ATPase activity. This was previously also shown by statistical analysis where fibre size was additionally taken into account [ 4 ]. Nifedipine was used as an antagonist in order to specifically block the dihydropyridine receptors. Nifedipine is a dihydropyridine derivative that binds in a specific, stereoselective and saturable fashion to DHPR. The selective calcium channel inhibitor prevents the influx of extracellular calcium through the L-type calcium channel [ 15 ] and also has a clear effect on the contraction activity of the skeletal muscle fibre. The effects of nifedipine on depolarization-induced force responses are inhibitory and dose dependent [ 16 ]. As the half life of the blocker is 2 – 6 h, there is enough time to perform reliable measurements. By blocking the channel with nifedipine, a clear selective decrease in the contraction force of the different muscles studied was observed (Fig. 3 ). The inhibition percentages of RF and GLU were, however, significantly higher as compared to that of GAS. This data indicates that the same concentration of blocker causes a different response between different muscles. A similar variability is noted when compared to the cell type composition of the muscles. The muscles with high IIA fibre type content have strongly reduced contraction force as a result of addition of a calcium channel blocker. On the other hand, the effect of a blocker on the muscle with a lower IIA type content is much weaker. The findings are attributable to the varying densities of dihydropyridine receptors in muscles. In GAS, the amount of DHPRs is reduced, likely due to the low IIA fibre type content. Thus the blocking effect of a receptor antagonist is weaker as compared to the muscles with a higher IIA content. Furthermore, the response of GAS and GLU to the gradual saturation of DHPRs with nifedipine molecules is different (Fig. 4 ). Consistently, the inhibition percentage of the contraction force of GAS decreased less as compared to GLU. In addition, a constant level of the contraction force was reached sooner in GAS indicating a complete saturation of the receptors available. There are several examples of the divergent behaviour of the dihydropyridine receptor in skeletal muscle. First, dihydropyridines are shown to have both stimulatory [ 17 ] and inhibitory [ 18 ] effects on excitation-contraction coupling. Furthermore, unlike in cardiac muscle, calcium release from SR does not require inward current, and as yet is induced by blockade of dihydropyridine receptors [ 19 ]. However, it is clear that DHPRs are essential in excitation-contraction coupling since animals with mdg/mdg mutation, which results in a lack of receptor proteins, die at birth because of paralysis of the respiratory muscles [ 20 ]. On the other hand, when the α 1 -subunit is introduced into the nuclei of the dysgenic myotubes, some cells contract upon an electrical stimulation. Despite of this, the recovered influx of calcium ions is not necessary for the contraction since the cells are contracting also even if the current is blocked with cadmium. Hence calcium antagonist drugs seem to have very few pharmacologically relevant actions on skeletal muscle as observed also by Walsh et al [ 21 ]. The role of inward current is still an open question although it has been suggested that the current is needed to maintain the calcium stores inside the cell [ 5 ] or for the conditions of activeness of the voltage sensor [ 22 ]. In the present study we report one plausible explanation for the large variation of the results observed in the behaviour of the L-type calcium channels in skeletal muscle. The uneven density distribution of dihydropyridine receptors indicates a difference in E-C coupling machinery between muscle fibre types. In this study, we showed that the difference is also detectable in the contraction forces between muscles of different cell type composition. Although nifedipine specifically blocks the dihydropyridine receptor, there could also be other differences in addition to the density of receptors between the muscles causing the difference in the contraction forces. A larger number of different muscles and developmental stages might clarify the differences in the present results. Goodman et al [ 3 ] provided the first evidence that E-C coupling characteristics are related to fibre types based on myosin heavy chain. By measuring depolarization-induced force responses of skinned single fibres of rat, they concluded that the optimum force production of a skeletal muscle is related to its MHC isoform composition. Although we found no published data on the E-C coupling phenotype of MHC IIa isoform, the results from previous studies suggest that the parameters describing force and velocity properties of a single muscle fibre are significantly higher in the fast, type IIA and IIB, fibres than in slow, type I fibres. Conclusions Taken together, our results obtained from three different muscles confirmed that the expression of dihydropyridine receptor is a fibre type specific character. In addition, the present data indicate that the density of DHPRs is one important factor influencing the overall contractile properties of the muscle. Furthermore, the results of our experiments point out that when determining the physiological relevance of DHPRs, it is necessary to compare histochemistry and protein analysis to relevant functional properties such as contraction of the muscle. Moreover, it is noted, that differences between developing and adult type of muscle cells may emerge due to the differentiation of phenotypes of muscle fibres. In the future it might be useful to examine the correlation from a larger number of different muscles, and from muscles of different developmental stage. Methods Electrophoretic separation of myosin heavy chain isoforms M. rectus femoris (N = 6), m. gastrocnemius (N = 6) and m. gluteus maximus (N = 6) from adult mice (strain CD-1) were removed after the animals were killed by paracervical dislocation in accordance with the Animal Ethics Committee of the University of Oulu (licence no. 046/03). The muscles were homogenized in 6 vol of homogenization buffer [62.5 mM Tris-HCl, pH 6.8] and boiled in sample buffer as previously described for 5 min at a final protein concentration of 0.5 mg ml -1 [ 23 ]. Total protein was assayed according to the method of Bradford [ 24 ]. Myosin heavy chain isoforms were separated by gradient (5–8 %) sodiumdodecylsulfate polyacrylamide gel electrophoresis performed at 5°C for 23 h (120 V constant voltage). The gels were stained with Coomassie Brilliant Blue. The stained gels were scanned and the separated protein bands were analysed with FluorS MultiImager program (Bio-Rad, USA). Fibre typing Serial cross-sections, 8 μm thick, were cut on a cryostat microtome at -25°C, mounted on cover slips and stained for myosinATPase (mATPase) with acid preincubation according to Hämäläinen and Pette [ 25 ]. The sections were preincubated at room temperature for 7 min in sodium acetate (54.3 mM) – sodium barbital (32.6 mM) solution adjusted with HCl to pH 4.6. After washing with CaCl 2 (18 mM) and Tris-HCl (199 mM) the sections were incubated in substrate solution (4.5 mM ATP, 19.5 mM CaCl 2 , 116 mM 2-amino-2-methyl-1-propanol; pH 9.4) at room temperature for 45 min. After incubations in 11 mM CaCl 2 , 2% CoCl 2 , and 10 mM sodium barbital, the colour was developed in 2% (v/v) ammonium sulphide for 45 s. After washing with distilled water, the sections were dehydrated in ethanol, cleared in xylene and mounted with DPX. An alkaline preincubation in a solution containing 34 mM 2-amino-2-methyl-1-propanol, 120 mM CaCl 2 , adjusted to pH 10.3 with HCl was also used as previously described by Guth and Samaha [ 26 ]. After preincubation the sections were processed as above. The percentage of each fibre type in different muscles was calculated with the use of the LSM 5 PASCAL software 3.2 (Leo, Germany) for analyzing images. Fluorescence staining For fluorescence labelling, RF muscle from mouse was cut into 8 μm cryostat sections, dried for 30 min and treated as previously described by Mänttäri et al [ 4 ]. The concentration of the high affinity (-)-enantiomer of dihydropyridine was 20 nM. Control samples were preincubated with 10 μM nifedipine in the phosphate buffer for 10 min prior to the addition of dihydropyridine conjugate. Force measurements Adult mice (weighing 23 – 45 g) were killed by paracervical dislocation, and the pelvic region and right hind limb were skinned. The muscle of interest (m. rectus femoris, m. gastrocnemius or m. gluteus maximus) was dissected out and suspended between a fixed clamp at the base of an organ bath and a Grass FTO3C (USA) force-displacement transducer. Muscles were maintained in an oxygenated (95% O 2 – 5% CO 2 ) physiological saline solution (+37°C, pH 7.4) containing 137.0 mM NaCl, 2.7 mM KCl, 1.0 mM MgSO 4 · 7H 2 O, 1.8 mM CaCl 2 , 0.4 mM NaH 2 PO 4 , 12 mM NaHCO 3 and 5.5 mM glucose. After an equilibration period of 2 min, the muscles were supramaximally stimulated using steel electrode, pulses of 1 ms duration at 80 V (model S44, Grass Instrument Co.) and muscle length selected to elicit maximal twitch force. The maximum force of the muscle was measured in series of three stimulus impulses with one minute equilibration time between the stimulations. Recordings of transducer output were A/D converted and collected on a computer at a sample frequency of 1000 Hz. In order to examine the role of a specific, L-type calcium channel blocker, the muscles first measured in saline solution were mounted in a bath containing 1 μM nifedipine [1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester], equilibrated for 2 min, and assessed as described above i.e. the muscle was a control in itself. In addition, series of measurements were made with m. gastrocnemius (N = 6) and m. gluteus (N = 6) using nifedipine concentration range of 2 – 30 μM. The concentration of the drug was progressively increased by adding the nifedipine solution to the saline solution bath. During the experiment, the nifedipine solutions were maintained in dark to prevent photo-bleaching. To exclude the possibility of fatigue caused by successive stimulations, the protocol control measurement was performed similarly devoid of nifedipine. Statistical analysis The statistical significance of differences between means of contraction forces before and after the addition of calcium channel blocker was evaluated by paired samples t-test. The difference in parameters between the different types of muscle were analysed with independent samples t-test for equality of means. A P value of < 0.05 was accepted as indicative of a significant difference between the two sets of observations. The significance of differences between the muscles was evaluated with one way analysis of variance (normality test passed). The significant difference was stated as mentioned above. The values are presented as the means ± SE. All the statistical analyses were performed with the SPSS for Windows software. List of abbreviations used DHP; dihydropyridine DHPR; dihydropyridine receptor E-C coupling; excitation-contraction coupling FOG; fast oxidative glycolytic GAS; musculus gastrocnemius GLU; musculus gluteus maximus mATPase; myosin adenosine triphosphatase MHC; myosin heavy chain RF; musculus rectus femoris SDS-PAGE; sodium dodecyl sulphate polyacrylamide gel electrophoresis SR; sarcoplasmatic reticulum T-tubule; transverse tubule IIa, IIb, IId; myosin heavy chain type IIA, IIB, IID; fibre type Authors' contributions SM carried out most of the experiments, assisted in interpretation of results, and participated in writing the manuscript. MJ conceived of the study, participated in its design and coordination, and participated in writing the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC550649.xml
340952	Design and Diversity in Bacterial Chemotaxis: A Comparative Study in Escherichia coli and Bacillus subtilis	Comparable processes in different species often involve homologous genes. One question is whether the network structure, in particular the feedback control structure, is also conserved. The bacterial chemotaxis pathways in E. coli and B. subtilis both regulate the same task, namely, excitation and adaptation to environmental signals. Both pathways employ many orthologous genes. Yet how these orthologs contribute to network function in each organism is different. To investigate this problem, we propose what is to our knowledge the first computational model for B. subtilis chemotaxis and compare it to previously published models for chemotaxis in E. coli . The models reveal that the core control strategy for signal processing is the same in both organisms, though in B. subtilis there are two additional feedback loops that provide an additional layer of regulation and robustness. Furthermore, the network structures are different despite the similarity of the proteins in each organism. These results demonstrate the limitations of pathway inferences based solely on homology and suggest that the control strategy is an evolutionarily conserved property.	Introduction Chemotaxis is the process by which motile bacteria sense changes in their chemical environment and move to more favorable conditions ( Bren and Eisenbach 2000 ). In peritrichously flagellated bacteria such as Escherichia coli and Bacillus subtilis , swimming alternates between smooth runs and reorientating tumbles. Smooth runs require that the flagellar motors spin counterclockwise, whereas tumbles result from clockwise spins. Bacteria follow a random walk that is biased in the presence of gradients of attractants and repellents by alternating the frequency of runs and tumble. Owing to their small size, most bacteria are unable to sense chemical gradients across the length of their body. Rather, they respond only to temporal changes. In particular, their stimulated response always returns to prestimulus levels despite the sustained presence of attractants or repellents. Sensory adaptation involves a rudimentary form of memory that allows bacteria to compare their current and past environments. Bacteria regulate chemotaxis using a network of interacting proteins. The basic mechanism in flagellated bacteria involves receptor-mediated phosphorylation of a cytoplasmic protein (CheY) that binds to the flagellar motor and changes the spin direction ( Falke et al. 1997 ). This pathway is characterized best in the γ-proteobacteria— E. coli and Salmonella enterica serovar typhimurium . Even though less is known about chemotaxis in other species of bacteria, the evidence so far suggests that the pathways are mechanistically different despite the homology of the individual genes to their γ-proteobacteria counterparts. B. subtilis , Helicobacter pylori, Myxococcus xanthus, Rhodobacter sphaeriodes , and Sinorhizobium meliloti , for example, all use similar yet distinct set of pathway components to regulate chemotaxis ( Armitage and Schmitt 1997 ; Ward and Zusman 1999 ; Pittman et al. 2001 ; Sonenshein et al. 2002 ). E. coli and B. subtilis bias their motion towards favorable conditions with nearly identical behavior by adjusting the frequency of straight runs and reorienting tumbles. Both pathways (summarized in Figure 1 and Table 1) share five orthologous proteins with apparently identical biochemistry. How these individual orthologs contribute to the overall function, however, is different, as illustrated when synonymous orthologs are deleted in each organism. Deletion of the CheY response regulator causes E. coli to run exclusively and B. subtilis to tumble exclusively ( Bischoff et al. 1993 ). When the CheR methyltransferase is deleted in E. coli , the cells are incapable of tumbles and only run. Likewise, when the CheB methylesterase is deleted, E. coli cells are incapable of runs and only tumble. In B. subtilis , cells still run and tumble when either CheB or CheR is deleted, though they no longer precisely adapt ( Kirsch et al. 1993a , 1993b ). Remarkably, both genes complement in the heterologous host. Deletion of the CheW adaptor protein in E. coli results in a run-only phenotype, whereas there is no change in phenotype for the synonymous deletion in B. subtilis . When the genes involved in regulating methylation are deleted ( cheBR in E. coli and cheBCDR in B. subtilis ), E. coli does not adapt ( Segall et al. 1986 ), whereas B. subtilis either oscillates or partially adapts when exposed to attractants ( Kirby et al. 1999 ). These differences demonstrate that the pathways are different even though they involve homologous proteins. Figure 1 The Chemotaxis Pathways in E. coli and B. subtilis (A) E. coli . (B) B. subtilis . Both organisms respond to extracellular signals by regulating the activity of the CheA histidine kinase. CheA is coupled to transmembrane receptors (MCP) by an adaptor protein CheW. Chemoattractants, by binding the receptor, inhibit CheA in E. coli (red line) ( Borkovich et al. 1989 ) and stimulate CheA in B. subtilis (green line) ( Garrity and Ordal 1997 ). CheA phosphorylates CheY. Phosphorylated CheY binds to the flagellar motor and increases the frequency of tumbles in E. coli ( Cluzel et al. 2000 ) and runs in B. subtilis ( Bischoff et al. 1993 ). Phosphorylated CheY is also predicted to inhibit the receptor complex in B. subtilis (dashed line). Both organisms tune the sensitivity of CheA to ligands by reversibly methylating the receptors using the CheR methytransferase and CheB methylesterase ( Zimmer et al. 2000 ; Sourjik and Berg 2002b ). Phosphorylation of CheB by CheA increases its methylesterase activity nearly 100-fold ( Anand and Stock 2002 ). CheA activity is proportional to the degree of receptor methylation in E. coli . In B. subtilis , CheA activity depends on which residue is methylated, akin to a binary switch. E. coli possesses a phosphatase, CheZ, not present in B. subtilis , that enhances the rate of CheY dephosphorylation. B. subtilis possesses three chemotaxis proteins not found in E. coli : CheC, CheD, and CheV. CheC is a negative regulator of receptor methylation and homologous to the CheY-binding domain (P2) in CheA ( Rosario et al. 1995 ; Rosario and Ordal 1996 ). CheD is a positive regulator of receptor methylation and also deamidates specific residues on the receptor ( Kristich and Ordal 2002 ). CheV is a CheW-response regulator fusion. CheV is functionally redundant to CheW and is predicted to negatively regulate receptor activity (dashed line) ( Rosario et al. 1994 ; Karatan et al. 2001 ). Table 1 Summary of Differences between E. coli and B. subtilis Chemotaxis To analyze and compare the two networks, we constructed mathematical models of both pathways. Numerous mathematical models exist for the chemotaxis pathway in E. coli ( Goldbeter and Koshland 1982 ; Asakura and Honda 1984 ; Knox et al. 1986 ; Bray et al. 1993 ; Bray and Bourret 1995 ; Hauri and Ross 1995 ; Barkai and Leibler 1997 ; Spiro et al. 1997 ; Morton-Firth et al. 1999 ), and we combined the models proposed by Barkai and Leibler (1997) and Sourjik and Berg (2002a ). For B. subtilis , we constructed a mathematical model that proposes an alternative mechanism for sensory excitation and adaptation. We validated the model against published data for B. subtilis chemotaxis. As there are fewer data concerning chemotaxis in B. subtilis , the model makes predictions regarding the function of the chemotaxis proteins CheC, CheD, and CheV not present in E. coli. Both models demonstrate how two divergent species mediate the same task using orthologous genes with different circuitry. Despite the differences, both pathways involve the same control strategy. The mathematical details of both models are described in Materials and Methods . Model Assumptions and Justification Both E. coli and B. subtilis regulate motility by controlling the phosphorylation of the CheY response regulator using the CheA histidine kinase. Phosphorylated CheY binds to the flagellar motor and increases the likelihood of reorientating tumbles in E. coli and straight runs in B. subtilis ( Bischoff et al. 1993 ). CheY is dephosphorylated by the CheZ phosphatase in E. coli. B. subtilis does not possess a homolog to the CheZ phosphatase. Instead, the motor switch protein FliY is the phosphatase for CheY in B. subtilis . CheA forms a complex with transmembrane receptors and CheW. When chemoattractants bind to the receptors, CheA is inhibited in E. coli and activated in B. subtilis . The net result is the same in both organisms: chemoattractants increase the likelihood of straight runs. Building on the success of the E. coli models ( Barkai and Leibler 1997 ; Morton-Firth et al. 1999 ), we employed a variant of the two-state model for receptor activation in B. subtilis . The two-state model treats the chemotaxis receptors, CheW, and CheA as a single entity and assumes the receptor complex adopts either an active or inactive comformation. Implicit in the two-state model is the assumption that the receptor complex is stable. The model assumes that the rate of CheA autophosphorylation is proportional to the average number of active receptor complexes in the cell. CheA, in turn, controls the rate of the phosphorylation for CheB, CheV, and CheY, as it is the phosphodonor. As the phosphorylation kinetics in B. subtilis have not been extensively investigated, the model uses the mechanism and parameters for phosphorylation cascade in E. coli proposed by Sourjik and Berg (2002a ). Both organisms respond and adapt to chemoattractants at comparable speeds ( Kirby et al. 1999 ; Sourjik and Berg 2002b ), so it is reasonable to assume that the phosphorylation rates are similar. The model assumes that the mechanism for CheV phosphorylation is the same as CheY and CheB. In E. coli , CheW regulates CheA activity in a biphasic manner ( Gegner et al. 1992 ). Ternary signaling complexes form when CheW joins receptor dimers with CheA dimers. The actual stoichiometry of the signaling complex is unknown, though it is known to form higher-order structures ( Stock and Da Re 1999 ). At low concentrations, the number of signaling complexes is proportional to the concentration of CheW. At higher concentrations, CheW inhibits the formation of ternary signaling complexes. Instead of ternary (active) complexes, partial (inactive) complexes of receptor–CheW and CheW–CheA form. Only at intermediate, stoichiometric concentrations of CheW do the majority of free receptors and CheA form active ternary complexes. In addition to CheW, chemotaxis in B. subtilis involves CheV, a CheW–response regulator fusion. CheV is functionally redundant to CheW: deletion of either gene has no visible effect on chemotaxis ( Rosario et al. 1994 ). Unlike CheW, the additional response regulator domain on CheV is necessary for proper function ( Karatan et al. 2001 ). We propose that CheV forms an additional layer of regulation in B. subtilis , where phosphorylation of the response regulator domain activates CheV. By regulating the number of active CheV molecules, B. subtilis could dynamically regulates the number of functional signaling complexes using a biphasic mechanism similar to CheW. The model simplifies this proposed mechanism for parsimony and assumes unphosphorylated CheV disrupts the receptor complex and inhibits the activation of CheA. This feedback mechanism proposes a role for CheV in addition to its functional redundancy to CheW. We note that H. pylori precisely adapts using a methylation-independent process involving three CheV paralogs ( Pittman et al. 2001 ), suggesting that perhaps it involves the same proposed CheV feedback mechanism for adaptation. B. subtilis also employs a methylation-independent chemotaxis mechanism; unlike E. coli , it still partially adapts to chemoattractants even when receptor methylation is disabled ( Kirsch et al. 1993a , 1993b ; Rosario et al. 1995 ; Rosario and Ordal 1996 ). The model assumes that phosphorylated CheY forms a negative feedback loop, where it inactivates CheA by binding to receptors. No such loop exists in E. coli . Experimental data for B. subtilis (discussed later) indicate that CheY interacts with the receptors. This model provides one possible feedback mechanism for methylation-independent chemotaxis. The other possibility is CheV. While either CheY or CheV is sufficient for methylation-independent chemotaxis, the model predicts that both feedback loops are necessary to generate the oscillations that are observed in the cheBCDR strains ( Kirby et al. 1999 ). The phosphorylation cascade is summarized in Figure 2 . Figure 2 Model for the Phosphorylation Cascade in B. subtilis The model assumes that the receptor complex (receptor, CheA, CheC, CheD, and CheW) exists either in an active ( T A ) or inactive ( T I ) state. Active receptors stimulate CheA. CheA phosphorylates CheB, CheV, and CheY. Phosphorylated CheY ( Y p ) binds the receptor and increases the likelihood a receptor adopts an inactive conformation (thick red line). Phosphorylated CheY also binds the flagellar motor ( M ). The motor switch enhances the rate of CheY dephosphorylation ( Szurmant et al. 2003 ). The model assumes that unphosphorylated CheV inhibits CheA by disrupting the receptor complex (thick blue line). In E. coli , CheA activity is roughly proportional to the number of methylated residues on the receptor ( Bornhorst and Falke 2001 ). E. coli adapts by altering the level of receptor methylation ( Goy et al. 1977 ). In B. subtilis , CheA activity depends on the specific residue methylated. In the model, we propose that methylation of residue E630 increases activity, whereas methylation of residue E637 decreases activity. The model is supported by the following experiments ( Zimmer et al. 2000 ). The amino acid substitution E630D, which renders the site permanently demethylated, decreases the activity of CheA, as inferred by analyzing the spin of the flagellar motor. Likewise, the substitution E637D increases the activity of CheA. In addition to residues E630 and E637, residue Q371 is also reversibly methylated. However, the substitution Q371D does not alter the activity or interfere with adaptation. As a result, we ignored it in the model. The model predicts that B. subtilis adapts to the addition of attractants by demethylating residue E630 and methylating residue E637. The reverse process is used to adapt to the loss of attractants. When B. subtilis is stimulated either by the addition or removal of attractants, the chemotaxis receptors are rapidly demethylated and then slowly remethylated ( Kirby et al. 1997 ). Cast in terms of the model, one residue is demethylated and then the other is methylated. As a comparison, the receptors in E. coli are methylated when the cells are exposed to attractants and demethylated when the attractants are removed. When the cheY gene is deleted in B. subtilis , a methylation pattern similar to E. coli is observed: the receptors are demethylated when the cells are exposed to attractants and methylated when the attractants are removed ( Kirby et al. 1999 ). These results demonstrate that CheY is necessary for normal patterns of methylation in B. subtilis . Similar behavior is observed when mutations are made to the active site of CheY ( Kirby et al. 1999 ) or when missense mutations are made to a small region on the C-terminus of the McpB receptor (C. J. Kristich, unpublished data). These results suggest that phosphorylated CheY interacts with the receptor to coordinate selective methylation. In the model ( Figure 3 ), we propose that CheY forms a switch for selective methylation. Residue E637 is preferentially methylated when phosphorylated CheY binds to the receptor. Otherwise, residue E630 is methylated. This proposed mechanism explains the mutant behavior: when the interaction between phosphorylated CheY and the receptor is disrupted, only residue E630 is methylated. As methylation of this residue increases the activity of the CheA kinase, we expect that residue E630 is demethylated when cells are exposed to attractants and methylated when the attractants are removed (as observed in cheY mutants). However in the mutant, there are no complementary changes at residue E637, as it cannot be methylated. Figure 3 Model for Selective Methylation in B. subtilis The model assumes that the receptor dimers exist in six different methylation states. The different methylation states are denoted by the variable T ij , where the index i denotes the methylation state of residue 630 and j denotes the state of residue 637. For example, T 20 denotes the concentration of dimers with both residues methylated at position 630 and none at position 637. For simplicity, the model assumes that at most two residues are methylated as additional states are superfluous. When receptors are methylated at residue 630, the signaling complex preferentially adopts an active conformation. When residue 637 is methylated, the signaling complex preferentially adopts an inactive conformation. When the dimers are partially methylated, the strength of activation or inhibition is attenuated. Selective methylation is coordinated by phosphorylated CheY ( Y p ). CheR methylates residue 637 when phosphorylated CheY is bound to the receptor and methylates residue 630 otherwise. As discussed previously, the model also predicts that the proposed interaction between phosphorylated CheY and the receptor forms a negative feedback loop that inhibits the CheA kinase in addition to its role in methylation. These two mechanisms form the following regulatory feedback loop. When there is an excess of phosphorylated CheY, CheA is inhibited and residue E637 is preferentially methylated (inhibiting residue). Likewise, when the majority of CheY is unphosphorylated, CheA is not repressed and residue E630 is preferentially methylated (activating residue). This feedback loop provides a regulatory mechanism for adaptation otherwise absent in B. subtilis . While in E. coli CheB phopshorylation is not necessary for adaptation ( Alon et al. 1999 ), it forms a negative feedback loop as the rate of demethylation—catalyzed by CheB—is proportional to the activity of CheA ( Anand and Stock 2002 ). This feedback loop likely controls the basal activity and the speed of response ( Hauri and Ross 1995 ). However, in B. subtilis , the receptors are demethylated in response to both positive and negative stimuli. It is implausible that CheB phosphorylation provides a regulatory mechanism for selective methylation and, based on the available data, CheY provides the logical alternative. cheC and cheD , chemotaxis genes present in B. subtilis and missing in E. coli , are not treated explicitly in the model. Mutations to either gene are modeled implicitly by perturbing the kinetic parameters governing CheA activation and selective methylation. CheC is homologous to the P2 domain of CheA and the N-terminal domain of FliM ( Kirby et al. 2001 ). Both domains bind CheY in E. coli . When CheC is deleted, the steady-state level of receptor methylation is roughly twice wild-type levels ( Rosario and Ordal 1996 ). When CheD is deleted, the receptors are unmethylated ( Rosario et al. 1995 ). Yeast two-hybrid experiments suggest that CheC and CheD interact with one another ( Rosario and Ordal 1996 ). Collectively, these results suggest that CheC and CheD coordinate CheY-dependent selective methylation by protecting one residue and exposing the other using phosphorylated CheY as the cue. In addition to its role in methylation, CheD deaminates glutamine residues on the receptors ( Kristich and Ordal 2002 ). As cheD mutants respond weakly to the addition of chemoattractants ( Kirby et al. 2001 ), we hypothesize that deamidation strengthens the coupling between the receptor and CheA kinase. Simple loss of methylation is insufficient to explain the phenomena, since unmethylated cheR mutants still respond strongly to chemoattractants ( Kirsch et al. 1993b ). We model deletions to CheD by decreasing the transition rate between active and inactive receptor complexes. Our justification, based on the model, is that the period of oscillations of flagellar rotation in the cheBCDR mutant is 100 s ( Kirby et al. 1999 ), far slower than the response in wild-type (less than 1 s). Our biological justification is that the CheD modifications strengthen the coupling between the receptors and CheA. Barkai and Leibler (1997 ) demonstrated that activity-dependent methylation is necessary for robust adaptation in E. coli chemotaxis. They propose that CheB demethylates only active receptors. Subsequent models, involving more detail, require that CheR methylates only inactive receptor ( Morton-Firth et al. 1999 ; Barkai et al. 2001 ; Mello and Tu 2003a ). Adaptation results by balancing the rates of methylation and demethylation at steady state. In the B. subtilis model, activity-dependent methylation is also necessary for robust adaptation, albeit in a different form. With selective methylation, one option is that CheB demethylates residue 630 when the receptor is active and residue 637 when it is inactive. No equivalent assumption is necessary for CheR. Other alternatives are possible, though this one was the simplest considered. How CheB distinguishes between active and inactive receptors is unknown. Phosphorylation is not sufficient: receptors are also demethylated when CheA is inhibited ( Kirby et al. 1997 ). The cue likely involves the same feedback loop regulating selective methylation: CheB binds residue 630 when phosphorylated CheY is bound to the receptor and binds residue 637 otherwise. In the present two-state model, however, this mechanism is not sufficient for robust adaptation. It is necessary to assume that CheB explicitly distinguishes between active and inactive receptors (as is the case with the E. coli models). Few kinetic measurements have been made for B. subtilis . On the one hand, we expect that the rates and concentrations are comparable to their E. coli counterparts, given that many B. subtilis chemotaxis proteins complement in E. coli . On the other hand, the additional feedback loops involving CheV and CheY could mask differences in the rates and concentrations between the two species. Unlike E. coli , many properties of the B. subtilis model, such as the steady-state bias and adaptation time, are insensitive to the kinetic parameters, suggesting that perhaps chemotaxis is more robust in B. subtilis than in E. coli . For lack of a better alternative, we used E. coli parameters for the B. subtilis model when available, as they produce results in the B. subtilis model consistent with experimental measurements. Many regulatory interactions proposed in B. subtilis model were inferred from mutants and lack explicit experimental confirmation. There are a number of experiments that could test the predictions made by the model, and we describe just a few. One experiment is to correlate receptor methylation with CheA activity in vitro using purified components ( Ninfa et al. 1991 ; Borkovich et al. 1992 ). This in vitro setup could also be used to test CheD; the model predicts that CheD enhances CheA activity by post-translationally modifying the receptors. Another experimental option for correlating receptor methylation with CheA is to fuse fluorescent proteins to FliY and CheY and use fluorescence resonance energy transfer to measure the relative concentration of phosphorylated CheY for different engineered methylation states in vivo ( Sourjik and Berg 2002b ). The in vitro setup using purified components could test the proposed regulatory interactions between CheY and the receptor. We could also test the predicted regulatory interactions involving CheV by measuring the stability of the ternary receptor complex (receptor, CheV, and CheA) for different concentrations of phosphorylated CheA or CheV. Another option is to compare the response to ligand for different cheV mutants (e.g., cheBCDR versus cheBCDRV ). Results Alternate Mechanisms for Adaptation Timecourse simulations of the models illustrate the process of adaptation in E. coli ( Figure 4 A) and B. subtilis ( Figure 4 B). Both models accurately reproduce the observed adaptation kinetics ( Segall et al. 1986 ; Kirby et al. 1999 ). Upon the addition of attractant, the CheA kinase is inhibited in E. coli and activated in B. subtilis . This change correlates with a rapid decrease in the concentration of phosphorylated CheY in E. coli ( Borkovich et al. 1989 ) and a rapid increase in B. subtilis ( Garrity and Ordal 1997 ). Both species adapt by changing the methylation state of their receptors. Whereas adaptation to attractants in E. coli is commensurate with an increase in receptor methylation, adaptation in B. subtilis is commensurate with the change in the relative state of receptor methylation. The average number of residues methylated at position 630 decreases and the average number at position 637 increases. The relative change in methylation in B. subtilis correlates with the absolute change in methylation in E. coli . Both organisms adapt to the loss of attractants by reversing the process. Figure 4 Simulation of Adaptation in E. coli and B. subtilis Attractant (10 μM) is added at 500 s and removed at 1,000 s. (A) Timecourse simulation of phosphorylated CheY (left) and receptor methylation (right) in E. coli . (B) Timecourse simulation of phosphorylated CheY (left) and receptor methylation (right) in B. subtilis . In both species, adaptation correlates with changes in receptor methylation. The concentration of phosphorylated CheB is proportional to the concentration of active receptors in E. coli and B. subtilis . This mechanism makes sense for E. coli , where CheB phosphorylation forms a negative feedback loop by de-methylating active receptors. However, it makes little sense in B. subtilis , where both active and inactive receptors are demethylated. Remarkably, however, experiments and simulation demonstrate that inactive receptors are demethylated just as efficiently as active receptors in B. subtilis , despite the fact that phosphorylation is necessary for CheB activity. What role phosphorylation of CheB plays in B. subtilis is unknown. We note that the homolog to CheB in Campylobacter jejuni lacks a response regulator domain. The B. subtilis model predicts that differential changes in methylation are symmetric. The increase in methylation at position 637 is matched by an equal decrease in methylation at position 630. These results predict that the average number of residues methylated is constant at all times. Experiments, however, paint a different picture ( Kirby et al. 1999 ). While the total level of methylation is constant at steady state, dynamic changes in differential methylation are not symmetric. Upon the addition or removal of attractants, there is a rapid decrease in receptor methylation proportional to the amount of attractant added or removed. This rapid decrease is followed by slow increase in receptor methylation. Despite considerable effort, we were unable to develop a robust model that captures this asymmetric behavior. Likely, there are additional mechanisms involved. The logical suspects are CheC and CheD. One hypothesis is that CheC and CheD form a switch, where CheC protects one residue and CheD exposes the other. In such a model, the rate of demethylation needs to be much faster than that predicted by the E. coli kinetic parameters. While conceptually appealing, we are currently unable to propose such a mechanism that robustly adapts. Further elucidation of CheC and CheD is necessary. The model in this case clearly points out deficiencies in our knowledge. Adaptation Involves Similar Regulatory Strategy The two-state model for chemotaxis in E. coli assumes that CheR ( R ) binds only inactive receptors ( T I ) and that phosphorylated CheB ( B P ) binds active receptors ( T A ). In a simplified version of the model ( Barkai and Leibler 1997 ), receptor methylation m is described by the differential equation where k B and k R are the rate constants and K B and K R are the Michaelis constants for receptor demethylation and methylation, respectively. We assume that the concentration of phosphorylated CheB is proportional to the concentration of active receptors. As argued previously by Barkai and Leibler (1997 ), the rates of receptor methylation and demethylation are, respectively, monotonically decreasing and increasing functions of receptor activity. As they are monotonic, the two rates intersect only once ( Figure 5 A). Therefore, Equation (1) admits a single steady-state activity. As the rates are functions of receptor activity and not ligand concentration, the model precisely adapts to all ligand concentrations. The model is also robust; the rates are monotonic for all choices of kinetic parameters. However, where they intersect depends on the choice of kinetic parameters. Adaptation is robust, but other properties of the network are not. Similar arguments extend to the full model ( Yi et al. 2000 ; Mello and Tu 2003a ). Figure 5 Graphical Illustration of Mechanism for Robust Adaptation (A) Qualitative relationship among receptor activity, methylation, and demethylation in E. coli . The rate of demethylation is proportional to the number of active receptors, and the rate of methylation is inversely proportional to the number of active receptors. The system reaches steady state only when the two solid lines cross. As the rate of methylation decreases monotonically with receptor activity and the rate of demethylation increases monotonically with receptor activity, only one steady state is possible ( A* ) if the rates depend solely on receptor activity. The kinetic parameters change the slope of the curves, but not their monotonicity. Hence, adaptation is robust with respect to changes in the kinetic parameters. However, the point where they intersect does change with the parameters. (B) Qualitative relationship between receptor activity and the differential rate of methylation in B. subtilis . The net rate of methylation at residue 630 decreases monotonically with receptor activity, and the net rate of methylation at residue 637 increases monotonically with receptor activity. By the same arguments, only one steady state ( A* ) is possible and, hence, adaptation is robust in B. subtilis . The B. subtilis model assumes that methylation is coordinated by phosphorylated CheY ( Y p ) and that CheB demethylates active receptors ( T A ) at residue 630 and inactive receptors ( T I ) at residue 637. If we simplify the model, the concentrations of receptors with residues methylated at 630 ( m 630 )and 637 ( m 637 ) are described by the following two differential equations: where K Y is the Michaelis constant for phosphorylated CheY and the receptor. Subtracting Equation (3) from Equation (2) , we obtain the differential equation where Δ m = m 630 – m 637 . We assume the concentration Y P is proportional to the concentration of active receptors. The relative rate of methyation at residue 630 in Equation (2) is a monotonically decreasing function of receptor activity, and the relative rate of methylation at residue 637 in Equation (3) is an monotonically increasing function of receptor activity. By the same arguments used for the E. coli model, Equation (4) admits a single steady state ( Figure 5 B) and the system robustly adapts to all concentrations. The difference between the two species is how receptor methylation forms memory. E. coli forms memory using the absolute level of receptor methylation m , and B. subtilis forms memory using the differential level of receptor methylation Δ m . The structure of Equations (1) and (4) are identical. One rate—proportional to the number of inactive receptors—increases the memory term, while the other rate—proportional to the number of active receptors—decreases the memory term. Both processes reach steady state only when the memory matches the current state. The structural similarities imply that both species employ the same core control strategy. The decision process is the same; the difference is in how the process is instantiated. The analogy is to running the same program on two different kinds of computers: same software, different machine code. However, as the next section demonstrates, how susceptible these pathways are to perturbation is different, suggesting a distinct evolutionary advantage for each underlying design. Both mechanisms are robust; adaptation does not depend on the values of the kinetic parameters. Robust adaptation requires feedback with integral memory ( Yi et al. 2000 ). The same strategy is used in many engineering designs and, in fact, is a necessary component for robustness ( Wonham 1985 ). By including a memory term, a feedback controller is able to determine whether regulation is improving or degrading with time and dynamically compensate for changes in control. This similarity between biological and artificial controls suggests that engineering concepts such as integral feedback can be used to predict the regulatory structure of intracellular pathways as they direct model development and help exclude alternate models. As we have argued, the difference between the two organisms is how memory is stored using receptor methylation. From an engineering perspective, both designs— m and Δ m —are equivalent. Chemotaxis Is Robust Adaptation is robust in E. coli chemotaxis; changes in the relative level of CheR expression did not alter the ability of E. coli to adapt to attractants ( Alon et al. 1999 ). It has previously been argued that robustness is necessary for complex networks ( Gerhart and Kirschner 1997 ; Hartwell et al. 1999 ). The model predicts that adaptation is also robust in B. subtilis —not surprisingly, as we explicitly considered robustness in model development. While adaptation is robust in E. coli , other network properties, such as the steady-state levels of phosphorylated CheY and adaptation time, are not. As these properties also affect the ability of bacteria to respond effectively to their environment and find food sources, we hypothesize that the two additional feedback loops present in B. subtilis chemotaxis (see the blue and red thick lines in Figure 2 ) buffer against mutation and stochastic fluctuations in protein expression. As a comparison, we plotted the steady-state levels of CheY phosphorylation and adaptation time as a function of CheB and CheR concentrations ( Figure 6 ). Figure 6 demonstrates that both properties in E. coli are sensitive to the concentrations of CheB and CheR. These predictions are consistent with experimental results ( Alon et al. 1999 ). The B. subtilis model, on the other hand, predicts that the steady-state level of CheY phosphorylation is insensitive to the concentrations of CheB and CheR and that the adaptation time is insensitive to the concentration of CheR. These results are also consistent with experimental data, as deletions to either CheB or CheR do not change the network behavior in B. subtilis as strongly as they do in E. coli ( Kirsch et al. 1993a , 1993b). Figure 6 Sensitivity to Parameters in E. coli and B. subtilis (A) E. coli . (B) B. subtilis . The top figures are plots of the steady-state concentration of phosphorylated CheY as a function of CheB and CheR concentrations. The bottom figures are plots of the adaptation time as a function of CheB and CheR concentrations. Adaptation time is defined as the length of time from the peak concentration in phosphorylated CheY ( Y p ) to within 5% of the steady-state concentration after the addition of attractant (10 μM). For all the concentrations considered, both models precisely adapt. While adaptation is a necessary component of chemotaxis, there are other design requirements of equal importance. One is positioning the concentration of phosphorylated CheY in a narrow functional range. The flagellar motor is exquisitely sensitive to changes in the concentration of phosphorylated CheY ( Cluzel et al. 2000 ). Simulations of the models suggest that the steady-state concentration of phosphorylated CheY in B. subtilis , unlike E. coli , is robust to changes in the relative level of CheR expression ( Figure 6 ). As the B. subtilis pathway is more complex than that of E. coli , the robust positioning of phosphorylated CheY provides one possible benefit to offset the evolutionary cost associated with the additional complexity. Obviously, both organisms inhabit different ecological niches (colon and gut versus soil) and, as a result, are subject to different selective pressures, so it is difficult to explain their differences without further investigating the role of their environment. There is also the issue of sensitivity; E. coli is able to sense gradients in concentrations spanning five orders of magnitude. As formulated, both models fail to capture this observed behavior. Other mechanisms, such as receptor clustering ( Maddock and Shapiro 1993 ; Bray et al. 1998 ) and interactions between heterogeneous receptors ( Mello and Tu 2003b ), are needed to explain this sensitivity in E. coli . Experimental data suggest that the same mechanisms are involved in B. subtilis ( Kirby et al. 2000 ; Zimmer et al. 2002 ). Methylation-Independent Chemotaxis In the absence of CheR and CheB, computer simulations, consistent with experiments ( Kirsch et al. 1993a , 1993b ), demonstrate that B. subtilis partially adapts in response to the addition of chemoattractants (data not shown). The results are similar when either gene is deleted. A subpopulation (60%) of B. subtilis cheBCDR cells oscillates when stimulated with chemoattractants ( Kirby et al. 1999 ). To model this behavior, we reduced the rate of transition between active and inactive receptor complexes by a factor of 500. This change produced a relaxation oscillator with a period of roughly 100 s that is observed experimentally ( Figure 7 A). Wild-type cells respond in less than 1 s to attractants, thereby suggesting that the rate of signaling is slower in the mutant. We needed therefore to adjust the model to account for the relatively long period in the mutants. cheD mutants weakly respond to chemoattractants, suggesting that the coupling between the receptor and kinase is attenuated. These results suggest that CheD, which deaminates glutamine residues on the receptors ( Kristich and Ordal 2002 ), enhances the coupling in the signaling complex. Figure 7 Oscillations and Methylation-Independent Chemotaxis (A) Timecourse simulation of cheBCDR strain in B. subtilis subject to the addition of attractants (100 μM) at 200 s and the removal at 500 s. Concentration of CheV was set at 8 nM. (B) Timecourse simulation of the cheBCDR strain in B. subtilis subject to the addition of attractants (100 μM) at 200 s and the removal at 500 s, where the concentration of CheV is halved (4 nM). Oscillations are very sensitive to the choice of kinetic parameters. Experiments indicate that only a fraction of the cheBCDR mutants oscillate (60%). The remaining cells partially adapt to the addition of attractants ( Kirby et al. 1999 ). We propose that the differences arise from stochastic variations in protein concentrations. In our simulations, we transition between the two phenotypes by adjusting the concentration of CheV by a factor of 2 ( Figure 7 B). A similar change has no effect in simulated wild-type stains, consistent with the fact that experimental deletions of CheV do not produce a detectable phenotype. Chemical oscillations typically arise from the interplay of positive and negative feedback loops ( Ferrell 2002 ; Tyson et al. 2003 ). The model proposes that CheV and CheY form these feedback loops. There is no evidence to suggest that other feedback loops exist, as the remaining regulatory proteins are not present in the oscillating strain. The model predicts that CheV inhibition produces a positive feedback loop. Unphosphorylated CheV inhibits CheA activation (see the blue thick line in Figure 2 ). As the concentration of phosphorylated CheV increases, the inhibition of CheA decreases, as there is less unphosphorylated CheV. Less inhibition leads to more phosphorylated CheV, and the cycle repeats itself. The net result is a positive feedback loop. This positive feedback loop forms a hysteresis: the kinase still remains active after the attractant is removed. Hysteresis is a common cause of oscillations in signal transduction cascades, as it results in two stable steady states: one where the concentration of phopshorylated CheY is high and the other where the concentration is low ( Ferrell 2002 ). When this hysteresis is coupled with negative feedback by CheY, the pathway oscillates as the negative feedback loop drives the pathway away from the high steady state towards the low steady state and then the low towards the high. The hysteresis disappears when the model accounts for CheD owing to the associated change in the kinetics. Even in the model for wild-type B. subtilis , the CheV positive feedback loop increases the sensitivity of the signaling response to chemoattractants. These predictions assign another possible function to CheV distinct from CheW. CheV is present in many motile species of bacteria, including coliform bacteria such as S. typhimurium. CheY Feedback Is a Relic of Vestigial Chemotaxis Pathway We speculate that CheY feedback is a relic of a primitive chemotaxis pathway. It is unlikely that bacteria started with all of the necessary chemotaxis genes from the outset, but rather evolved or acquired methylation later ( Boyd and Simon 1982 ). The core pathway involving chemoreceptors, CheW, CheA, and CheY is present in all known species of motile bacteria. Homologs to the remaining chemotaxis genes are present in some species and absent in others, suggesting that they were subsequent innovations to the core pathway ( Table 2 ). If the core pathway was present before these additional genes were acquired, there would need to be some sort of stopgap regulation. As many of these additional genes are involved in methylation, we suspect that early pathways were regulated by a methylation-independent process. CheY feedback is the logical first step towards a functioning chemotaxis pathway, as it provides a mechanism for precise adaptation involving the core pathway without the need for additional genes ( Figure 8 ). The mechanism is not robust; the model is sensitive to the choice of parameters. If robustness is important for survival and environmental adaptation, perhaps then the methylation genes were acquired (CheB, CheC, CheD, and CheR) to address this flaw. Additional factors also favor the acquisition of methylation: methylation broadens the range of concentrations over which the bacteria are able to detect gradients and further implicates methylation as an evolutionary upgrade to primitive CheY feedback. Figure 8 CheY Feedback Is Sufficient for Precise Adaptation Timecourse simulation of model subject to the addition of attractants (10 μM) at 200 s and removal at 500 s. The model is described in Materials and Methods . Table 2 Distribution of Chemotaxis-Like Genes and Number of Paralogs for a Representative Set of Microbial Organisms Genes were determined either by annotation or simple BLAST searches. R. sphaeroides genes were taken from Porter and Armitage (2002 ). Some chemotaxis-like genes are not directly involved in motility, but are involved in other process, such as development ( Kirby and Zusman 2003 ). Other genes, in particular paralogs to CheY, may be false positives. For further information, including FASTA files and alignments, refer to http://genomics.lbl.gov/chris/chemotaxis/genes.html . This table updates a similar table presented by Armitage (1999 ) a There are additional orthologs to CheA, CheB, and CheR on the plasmids pSymA and pSymB b There is also a CheABR fusion c CheA is fused to CheY in C. jejuni and H. pylori d CheB lacks a response regulator domain in C. jejuni Discussion That the two pathways are different is not surprising, as E. coli and B. subtilis likely diverged over 1 billion years ago ( Kunst et al. 1997 ). That both organisms use homologous genes is also not surprising. Divergent species of bacteria likely tinker with a limited set of genes, as mutations that change regulatory interactions between genes are far more frequent than mutations that confer novel function ( Jacob 1977 ; Carroll et al. 2001 ). The genes may be similar, but how they interact with one another is different. In fact, other species of bacteria, each with their own idiosyncrasies, also have evolved novel chemotaxis pathways by tinkering with a small set of conserved genes and protein domains (see Table 2 ). The question then is whether other properties of the network, in addition to the genes, are conserved. The chemotaxis models for E. coli and B. subtilis indicate that the decision-making process is identical. The biochemistry is different, but the regulatory strategy is the same. Does this mean that regulation is conserved? Selective pressures likely constrain the evolution of most networks to ensure they function robustly despite intrinsic noise due to molecular fluctuations, stochastic gene expression, and mutation ( Hartwell et al. 1999 ; von Dassow et al. 2000 ). Consequently, regulation becomes an indirect object of selection. As diverse physiological processes have equivalent regulatory needs such as homeostasis and adaptation, the underlying pathways, based on this hypothesis, involve identical control strategies. Bacteria constantly prune their genome, removing redundant and nonessential genes ( Mira et al. 2001 ). As the chemotaxis pathways in E. coli and B. subtilis are functionally equivalent, it is not evident why chemotaxis is more complex in B. subtilis than in E. coli . One hypothesis is that the additional genes and feedback loops buffer against genetic mutation, though why B. subtilis is more robust is not clear. As both organisms inhabit different environments, the alternate designs and associated tradeoffs likely reflect niche adaptation. A similar hypothesis regarding the evolution of regulatory networks was proposed by Savageau (2001 ) in his demand theory for metabolism. As evident from bacterial chemotaxis, we cannot necessarily predict the structure and behavior of a network based on protein homology alone, as subtle differences in the proteins affect how they function in the network and with whom they interact. As these differences result from alternate regulatory interactions, comparing and analyzing these loops in divergent organisms provide insight regarding the properties and design of intracellular networks. By studying bacteria in different environments, we can learn how network structures evolve. By constructing a model of B. subtilis chemotaxis and comparing it to models of E. coli chemotaxis, we were able to explore two mechanisms for sensory adaptation involving homologous genes. These models enabled us to interpret a large class of data involving many different experimental conditions and mutants. The conclusion from this theoretical study is that both networks involve the same core control process, though the physical interactions and feedback loops that form this process are different. The implication is that we need to study the systematic properties of the homologous pathway in divergent organisms, rather than focusing exclusively on the individual genes. The hope is to understand the relative advantage and significance of each design and not exhaustively study each special case. Materials and Methods All simulations were performed in Matlab (Mathworks, Natick, Massachusetts, United States). Matlab m-files are available from http://genomics.lbl.gov/~chris/chemotaxis . E. coli chemotaxis model. The chemotaxis model combines the two-state model proposed for adaptation by Barkai and Leibler (1997 ), with the model for the phosphorylation cascade proposed by Sourjik and Berg (2002a ). The two-state model assumes that receptor complexes T exist in either an active ( T A ) or inactive ( T I ) state. Let T i denote the concentration of receptor complexes with i residues methylated and α i ( L ) denote the probability that the receptor complex T i is active when the concentration of chemoattractant is L . The concentration of active receptors is and the concentration of inactive receptors is For simplicity, we assumed that ligand binding is fast and employed the quasi-steady-state assumption. The probabilities α i ( L ) are given by the expression with these parameters: α 0 = 0; α 1 = 0.1; α 2 = 0.5; α 3 = 0.75; α 4 = 1; α L 0 = 0; α L 1 = 0; α L 2 = 0.1; α L 3 = 0.5; α L 4 = 1; K L = 10 μM ( Barkai and Leibler 1997 ). We modeled the phosphorylation cascade using the mechanism and parameters proposed by Sourjik and Berg (2002a ). We extended their model to include CheB phosphorylation. The parameters for CheB phosphorylation were inferred from the wild-type adaptation kinetics ( Sourjik and Berg 2002b ): The terms A and A p denote the concentrations of CheA and phosphorylated CheA, Y and Y p denote the concentrations of CheY and phosphorylated CheY, B and B p denote the concentrations of CheB and phosphorylated CheB, M denotes the concentration of FliM, and [ MY p ] denotes the concentration of phosphorylated CheY bound to FliM. We modeled receptor methylation using the mechanism proposed by Barkai and Leibler (1997 ), with the extensions proposed by Morton-Firth et al. (1999 ). For simplicity, we assume that the methylation reactions follow Michaelis–Menten kinetics. Similar results were obtained using mass action kinetics. In the Morton-Firth model, CheR binds only inactive receptors and phosphorylated CheB binds only active receptors. For the receptor T i , the rate of demethylation is r Bα i ( L ) T i and the rate of methylation is r B (1 – α i ( L )) T i , where and Note that the rate of methylation is proportional to concentration of inactive receptors (1 – α i ( L )) T i and the rate of demethylation is proportional to the concentration of active receptors α i ( L ) T i . A simple mass balance yields the following set of differential equations for the receptors: The parameters for the model are: k r = 0.255 s –1 ; K R = 0.251 nM; k B = 0.5 s –1 ; K B = 5.5 nM; A + A p = 5 nM; B + B p = 2 nM; Y + Y p + [ MY p ] = 17.9 nM; M + [ MY p ] = 5.8 nM; and T 0 + T 1 + T 2 + T 3 + T 4 = 5 nM ( Sourjik and Berg 2002a ). We note that the estimated concentrations for FliM and CheY were for fluorescent fusion proteins expressed from a plasmid and may be different from the wild-type concentrations. B. subtilis chemotaxis model. The B. subtilis model employs a variation of the two-state model proposed for E. coli . The model assumes that the receptor complex adopts either an active or inactive conformation. However, receptors can adopt one of four signaling states: either active, inactive, weakly active, or weakly inactive. In this regard, the model distinguishes between the signaling state of receptor complex and receptor, and it can be considered a heterogeneous two-state model ( Bornhorst and Falke 2003 ). Let T ij denote the concentration of receptor dimers with i residues at position 630 methylated and j residues at position 637 methylated. We assume that at most two residues on a dimer are methylated. Additional methylation states are superfluous. The concentration of (strongly) active receptors is given by the expression and the concentration of (strongly) inactive receptors is given by the expression where i ij is the probability that the receptor complex T ij adopts an inactive conformation. The concentration of weakly active receptors is given by the expression where β( L ) is the probability that a weakly active receptor adopts an active conformation. The concentration of weakly inactive receptors is given by the expression The physical picture is the following. Receptors can either activate or inactivate the CheA kinase. Receptor methylation increases the magnitude of activation or inactivation, likely by stabilizing the conformational change and the coupling between the receptor and kinase. When receptors are methylated (either at residue 630 or 637), the probability that they adopt a strong conformation increases. Unmethylated receptors always adopt a weak (active or inactive) conformation. These assumptions were necessary to construct a robust model. In the E. coli model, there are two boundary conditions: fully methylated receptors and unmethylated receptors. Furthermore, methylated receptors are active (α = 1) and unmethylated receptors are inactive (α = 0). In the B. subtilis model, there are four boundary conditions: T 20 , T 02 , T 11 , and T 00 . Furthermore, the methylated receptors T 11 and unmethylated receptors T 00 are partially active. We needed, therefore, to distinguish additional states to construct a robust model involving activity-dependent methylation. In a similar manner to the E. coli model, we assume that the kinetics for ligand binding are fast and employ the quasi-steady-state assumption for simplicity. The probabilities α ij ( L ) and i ij ( L ) are given by the expressions with these parameters: α 20 = 1; α 10 = 0.4, α 11 = 0.2; α 00 = α 01 = α 02 = 0; α 0 20 = 1; α 0 10 = 0.99, α 0 11 = 0.8; α 0 00 = α 0 01 = α 0 02 = 0; i 02 = 1; i 01 = 0.99, i 11 = 0.8; i 00 = i 10 = i 20 = 0; i 0 02 = 1; i 0 01 = 0.4, i 0 11 = 0.2; i 0 00 = i 0 10 = i 0 20 = 0; β = 0.2; β 0 = 0.8; K L = 10 μM. The parameters were inferred from tethering experiments, where the attractant asparagine is added and then removed in a flowcell containing wild-type cells and the rotation of the flagellar motor is observed ( Kirby et al. 1999 ) The model assumes that CheY negatively regulates CheA activity. The model assumes that only phosphorylated CheY ( Y p ) binds receptors. We model receptor binding with the following two differential equations: where [ T ] and [ TY p ] denote, respectively, the concentration of unbound and Y p -bound receptors. We assume that the fraction of active receptor complexes C A satisfies the following differential equation: where k A = 0.5 [ T ](1 + 10 T A + 0.1 T WA ) and k I = 0.5 [ TY p ](2 + 10 T I + 0.1 T WI ). The term C denotes the concentration of inactive receptor complexes. Evident from the expressions for k A and k I , weakly active and inactive receptors contribute less to the state of the receptor complex. The model for the phosphorylation cascade in B. subtilis is an extension of the model proposed for E. coli . The key differences are the addition of CheV and the loss of CheZ. We used a Michaelis–Menten-type expression to model inhibition of the CheA kinase by unphosphorylated CheV ( V ). There is no dedicated phosphatase for CheY in B. subtilis . However, the motor switch appears to enhance the CheY dephosphorylation when phosphorylated CheY is bound to the motor ( Szurmant et al. 2003 ). We assume the rate of CheY dephosphorylation increases when phosphorylated CheY is bound to the motor: As we lack kinetic parameters for B. subtilis , we used the parameters from the E. coli model when available. The parameters for CheV and CheY dephosphorylation were chosen so that the dynamics of the model were similar to those observed in tethering experiments involving wild-type bacteria and cheBCDR mutants ( Kirby et al. 1999 ). For simplicity, we used Michaelis–Menten kinetics to model the methylation reactions. Similar results were obtained using mass action kinetics. For the receptor T ij , the rate of demethylation for residue 630 is r Bα ij ( L ) T ij and the rate of demethylation for residue 637 is r B i ij ( L ) T ij , where The model assumes that only (strongly) active and inactive receptors are demethylated. The rate of demethylation for residue 630 is proportional to the concentration of (strongly) active receptors, and the rate for residue 637 is proportional to the concentration of (strongly) inactive receptors. The rate of methylation for residue 630 is r 1 R T ij and the rate for residue 637 is r 2 R T ij , where and Note that the rate of methylation for residue 637 is simply the rate of methylation times the probability that the receptor is bound with Y p and vice versa. A simple mass balance yields the following differential equation for the receptors: The parameters are the same as the E. coli model: k r = 0.255 s –1 ; K R = 0.251 nM; k B = 0.5 s –1 ; K B = 5.5 nM; A + A p = 5 nM; B + B p = 2 nM; Y + Y p + [ MY p ] + [ TY p ] = 17.9 nM; M + [ MY p ] = 5.8 nM; T 20 + T 10 + T 00 + T 01 + T 02 + T 11 = 5 nM; [ T ] + [ TY p ] = 5 nM. The model assumes that the concentration of CheV is 8 nM: V + V p = 8 nM. To model oscillations for the cheBCDR strain described in Figure 7 , we used the following differential equation to describe the fraction of active receptor complexes C A where k A = 0.001 T (1 + 0.1 T WA ) and k I = 0.001[ TY p ] (2 + 0.1 T WI ) with the initial condition T 00 = 5 nM. The concentrations of CheB and CheR were set to 0 to account for their deletion. The subpopulation that partially adapts was modeled by setting the concentration of CheV = 4 nM. In this formulation, receptors adopt either a weakly active or weakly inactive conformation. We also induced a timescale separation necessary for a relaxation oscillator by decreasing the transition rate between active and inactive receptor complexes by a factor of 500. This change produced oscillations with a period of 100 s. To model precise adaptation with simple negative feedback by CheY as described in Figure 8 , we used the following differential equation to describe the fraction of active receptor complexes C A : where k A = 0.1[ T ] T WA and k I = 0.1[ TY p ] T WI with the initial condition T 00 = 5 nM. The concentrations of CheB and CheR were set to 0. We also needed to change the model for receptor binding: where k A = 0.01/(10 + L ) + 0.036 L /(10 + L ). Supporting Information Accession Numbers The GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) accession numbers for the genes and gene products discussed in this paper are E. coli CheA (AAC74958) and B. subtilis CheA (CAB13516), E. coli CheB (AAC74953) and B. subtilis CheB (CAB13506), B. subtilis CheC (CAB13518), B. subtilis CheD (CAB13519), E. coli CheR (AAC74954) and B. subtilis CheR (CAB14188), B. subtilis CheV (CAB13274), E. coli CheW (AAC74957) and B. subtilis CheW (CAB13517), E. coli CheY (AAC74952) and B. subtilis CheY (CAB13506), E. coli CheZ (AAC74951), and B. subtilis FliY (CAB13505).	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC340952.xml
517502	Osteoporosis-related life habits and knowledge about osteoporosis among women in El Salvador: A cross-sectional study	Background Osteoporosis is a systemic skeletal disorder, characterized by reduced bone mass, deterioration of bone structure, increased bone fragility, and increased fracture risk. It is more frequent to find among women than men at a 4:1 ratio. Evidence suggests that to adopt changes on some life habits can prevent or delay development of osteoporosis. Several osteoporosis-risk factors have been confirmed in the US and western Europe, but in El Salvador there are neither reliable epidemiological statistics about this skeletal disorder nor studies addressing osteoporosis-risk factors in women. The aim of this study was to determinate the extent of osteoporosis knowledge, the levels of both daily calcium intake and weight-bearing physical activity, and the influence of several osteoporosis-risk factors on these variables in three age groups of Salvadorean women. Methods In this exploratory cross-sectional study, an osteoporosis knowledge assessment questionnaire incluiding a food frequency and a physical activity record section were used to collect data and it was delivered through a face-to-face interview. A convenience sample (n = 197) comprised of three groups of women aged 25–35 years, 36–49 years, and over 49 years was taken. Among-group comparisons of means were analyzed by two-way ANOVA. To determinate the overall influence of osteoporosis-risk factors, the multivariate analysis was used. Results Study results indicated that better educated women had more knowledge about osteoporosis than women with a low education level, regardless of age, even though this knowledge was rather fair. Older women got more weight-bearing physical activity at home and less at place of employment than reported by the younger women; however, neither group performed sufficient high-intensity WBPA to improve bone mass. Regardless of age, the most women consumed 60% or less than the Dietary Reference Intake of calcium and depend on household income, lactose intolerance and coffee rather than milk consumption. Conclusion In summary, the majority of women in this study have modest knowledge on osteoporosis. The knowledge base is not linked to preventive health habits, including sufficient calcium intake and performance of weight-bearing physical activities. They are thus at increased risk for low bone mass.	Background Osteoporosis is a systemic skeletal disorder, characterized by reduction of bone mass, deterioration of bone structure, increasing bone fragility, and increasing fracture risk [ 1 - 4 ]. It is more frequent among women than among men [ 5 , 6 ]. The development of low bone mass is typically asymptomatic, with many women reporting clinical manifestations including acute back pain, limited back mobility, fragility fractures (hip, vertebrae, proximal femur, distal radius, humerus, tibia), compression of midthoracic vertebrae and upper lumbar vertebrae, progressive deformation of the spinal column (cyphosis), reduced height, and radiculopathies [ 2 , 4 - 6 , 8 , 9 ]. Several risk factors for osteoporosis have been identified, these include female sex; Caucasian or Asiatic race; advancing age; family history of osteoporosis or fragility fractures; a low body mass index; menopause before age 45 years; prolonged amenorrhea unrelated to menopause; nulliparity; prolonged lactation; diet low in calcium and vitamin D; poor intestinal absorption of calcium; lactose intolerance; excessive caffeine or alcohol consumption; smoking; sedentary lifestyle; and prolonged treatment with thyroid hormones, glucocorticoids (e.g. cortisone), anticonvulsants, aluminum antiacids, and use of anticoagulants [ 1 , 3 - 6 , 8 ]. Approximately 20% of bone mass is genetically determined; however, the risk of osteoporosis can be reduced by optimizing bone mass increasing during youth, conserving bone mass during adulthood, and minimizing bone mass loss during advancing age [ 5 - 7 ]. Among most important preventive habits are a) weight-bearing exercise (e.g. going up and down stairs, jogging, aerobics, swimming, and isometrics; at least 30 minutes daily), b) diet or supplements containing adequate levels of calcium and vitamin D, and c) absence or cessation of smoking and no greater than moderate alcohol and/or caffeine consumption [ 5 - 10 ]. A study on US women aged over 25 years found that knowledge about osteoporosis was limited, irrespective of age [ 11 ]. Calcium intake was sufficient in most cases, but the amount and type of physical activity was inadequate to achieve enhanced bone mass; most women in this study performed some physical activity in the course of paid work or housework, but did not admit to systematic daily exercise. Other study on Caucasian and African-American women found that most of them had heard about osteoporosis, but few women got both adequate exercise and the recommended intake of calcium per day [ 12 ]. Asian women in Australia also had lower calcium intake (< 800 mg/day) and their knowledge about osteoporosis was limited [ 13 ]. There have been three related studies reported in Hispanic women: a study performed in Mexico [ 14 ], and two studies on women of Hispanic origin in the US [ 15 , 16 ]. The Mexican study considered women aged 50 – 59 years, and focused on knowledge about menopause and risk associated with premature menopause. About 90% of subjects were aware of the relationship between menopause and osteoporosis, but most subjects had little knowledge of other risk factors, and incorporated life habits that clearly increased osteoporosis risk. The studies on women of Hispanic origin in the US have yielded somewhat contradictory results. The first of these found that more than 37% of women had adequate preventive habits including the taking of calcium supplements and the performance of regular exercise; much of this was attributed to prior health education, knowledge about osteoporosis, bone-mass evaluation offered by healthcare services, and medical advice [ 15 ]. The second study considered both Hispanic and African-American women, and found that most women in both groups had a poor knowledge of behaviors that promote and maintain bone mass [ 16 ]. Notably, less than 50% of women performed regular physical exercise, and less than 10% had adequate calcium intake. At present, the Salvadorean Public Health and Social Assistance Ministry does not maintain a specific record of this disease in adults, and most cases are likely classified as dorsalgy, that includes several musculoskeletal diseases such as radiculopathies, cervicalgy, lumbalgy, sciatica, spinal derived pain, and unspecified back pain [ 17 ]. This reflects the fact that osteoporosis may be reported as dorsalgy. According to this report from El Salvador, in 2001 dorsalgy was the seventh most frequent cause of morbidity in women aged 50 – 59 years attending outpatient clinics, with an incidence of 3,983 new cases per 100,000 inhabitants and a total of 8,989 consultations (first-time or subsequent); in women aged 60 years or more, there were 9,754 consultations for dorsalgy in 2001. Bearing in mind the lack of reliable epidemiological data, the present study wished to investigate osteoporosis-related life habits (including exercise and calcium intake) and knowledge about osteoporosis among Salvadorean women aged ≥ 25 years. We investigated possible relationships of these variables with age, educational level, household income, family history of osteoporosis, menopause before age 45 years, fecundity (children per woman), lactose intolerance, caffeine consumption, low in calcium diet, and use of aluminum antiacids. Methods Study design and sampling This was an exploratory study with a cross-sectional design performed between May and September 2003, and it was used to survey a convenience sample comprised of 197 women (73 aged 25 – 35 years, 74 aged 36 – 49 years, and 50 aged over 49 years) from urban areas within 6 main municipalities in El Salvador. All subjects were randomly sampled and recruited by personal contact through to visit homes, churches, schools, primary healthcare centers, hospitals, supermarkets, shopping centers, and parks. It was not necessary to obtain any proper informed consent from interviewed women. The participation rate of women was 87.5% (197 out of 225). We selected three age groups of women attending following criterions: women aged 25 – 35 years are at the period when higher peak of bone mass is reached [ 3 ]; women aged 36 – 49 years are at a period around menopause when bones undergo a slow mineral density loss [ 3 ]; finally, most women aged over 49 years have undergone menopause, when osteoporosis clinical manifestations may begin to show [ 3 ]. Data collection and instruments Data were obtained at the time of interview, which were performed by a member of the research team. Prior training in interview techniques was obtained for study. Each staff interviewer subsequently participated in several practice sessions, with an evaluation component to confirm accurate transcription of responses, and comparison and adjustment to ensure good inter-interviewer concordance. The data collection instrument incorporated a personal interview guide comprising five sections, validated through a prior pilot study performed in 12 women (4 in each age group). Among the demographic information was household income. It was classified by the criteria of the Salvadorean Economy Ministry as "below poverty line" if monthly income was below the cost of two basic shopping baskets (i.e. < $254), and otherwise as "above poverty line" [ 18 ]. The second survey section generated information on family history of osteoporosis, and asked about the use of aluminum antiacids. Section 3 addressed weight-bearing physical activity (e.g. walking, standing, climbing and descending stairs) both at place of employment and at home, and included questions on weight-bearing exercises including jogging, swimming, aerobics, and isometrics. This survey section was adopted from a previous study [ 11 ]. A test/ re-test procedure was administered a week apart on pilot study subjects to calculate reliability of next questionnaire sections. Test/re-test correlation was 0.67 for physical activity section. Section 4 produced information on diet: for each of a list of 31 dietary and non-dietary items (including dairy products, baked products, meat, vegetables, fruits, and calcium supplements), the subjects were asked to estimate frequency of consumption (daily, weekly, fortnightly, or monthly). Similar checklists have been used in related previous studies [ 19 ]. Given the high coffee consumption in Central America, the subjects were asked about their degree of coffee consumption. Test/re-test correlation was 0.72 for dietary and non dietary calcium intake section. Final section (5) comprised eight open questions designed to assess nine knowledge dimensions about osteoporosis in subjects. The nine knowledge dimensions regarding osteoporosis, their specific questions and corresponding scores (from 0 to 42) are shown in Table 1 . These questions served as a backbone for the interview and they were similar to other set used in a previous study [ 11 ]. Test/re-test correlation was 0.59 for osteoporosis knowledge questionnaire section. Data analysis Each subject's responses to the questions in the fifth section were analyzed by the principal researcher, who compared the answers with a semantic map (Fig. 1 ) developed by the authors on the basis of findings from previous studies on osteoporosis [ 1 , 2 , 5 - 7 ]. This approach allowed for quantification on knowledge about osteoporosis so: 5 points assigned for responses denoting knowledge of three or more concepts (each node in semantic map represents a concept), 3 points for responses denoting knowledge of 2 concepts, and 2 points for responses denoting knowledge about one out of osteoporosis-related concepts as reported previously [ 11 ]; except for osteoporosis information source dimension since its sub-score depended on number of information sources that women were able to mention, so that they only got one point per each source up to a maximum of 5 points. The total score achievable was 42 points. The total number of hours of weight-bearing physical activity was estimated by adding the subtotals for activities at home, activities at worksite, and that of weight-bearing exercises. Total calcium intake was estimated by adding the estimated subtotals for consumed dairy products, other dietary intake, and calcium supplements, including calcium content per portion × the number of portions per day. Obtained values were compared with reference values [ 20 ]. Data are cited in the text as means ± standard errors. For variables with homogeneous variance, means were compared by two-way analysis of variance and by Tukey tests for pairwise comparisons. For variables with non-homogenous variance, data were compared by the Kruskal-Wallis test (ANOVA on ranks) followed by Dunn's test for pairwise comparisons. These analyses were performed using SigmaStat version 2.03. Correlation analysis was used to determine relationships between knowledge scores and WBPA including exercise and calcium intake. To develop predictive models, we used a multivariate analysis based on multiple linear regression as contained in the program AMOS version 5 (Small Waters Corp.). Results Characteristics of the interviewed women The participant characteristics of each age group are shown in Table 2 . Frequency of women with none or only a primary education increased with age; conversely, frequency of women with secondary or higher education decreased with age. Household income reported that most women (> 61%) were below the poverty line; although the number of women living on poverty conditions fluctuated with age. The frequency of nulliparity among interviewed women declined with age, whereas both the frequency of parity among interviewed women and the fecundity rate (children per woman) increased with age as anticipated. In all age groups, more than 44% of interviewed women reported a family history of osteoporosis, which included extended family: grandmothers, mothers, aunts, elder sisters, and cousins. Median, mean (SEM), and range of age of menopause in interviewed women were: 44, 42.3 (± 0.8), and 27–53 years, respectively. About 20% of interviewed women had lactose intolerance or ingested aluminum antiacids, both of these characteristics were unrelated of age. In addition, most women (> 69%) were coffee-consumers and the consumption of this drink increased with age. Knowledge about osteoporosis The sub-scores per knowledge dimensions about osteoporosis and age groups are shown in Table 3 . The total scores of knowledge regarding osteoporosis were similar in the three age groups of women (median 14 in 25- to- 35-years; 17 in 36- to 49-years; 15 in over 49 years). Most of interviewed women (75%) had enough knowledge about osteoporosis regarding the concept of disorder and its risk factors, sex-related factor, and prevention behaviours, irrespective of age (Table 3 ). Conversely, these women got less knowledge scores for diagnosis and treatment of osteoporosis than other examined dimensions. In all three age groups, women with secondary or higher education obtained significantly higher total knowledge scores ( F = 22.46, p < 0.001) than women with lower educational level (Fig. 2 ). There was not a significant relationship between age and educational level ( F = 1.38, p = 0.223). Only 5% of the women with higher education obtained total scores of less than 12 points, and 75% of them obtained total scores of more than 25 (table 3 ). Multivariate analysis (factors age, educational level, family history of osteoporosis, household income, early menopause, fecundity) explained only about 37% of variance in total score (R 2 = 0.367, p < 0.001) (Fig. 3 ). The most meaningful predictors of total score were educational level (R = 0.47, p < 0.001), household income (R = 0.18, p = 0.015), and early menopause (R = 0.18, p = 0.003). Physical activities The amount of weight-bearing physical activity was similar in the three age groups (mean 8.7 ± 0.5 hours/day in women 25- to 35 years; 8.9 ± 0.5 hours/day in those 36- to 49 years; 8.3 ± 0.6 hours/day in those over 49 years). Analysis of variance with factors of age and family history of osteoporosis indicated that neither factor produced a significant effect (F = 0.23, p = 0.794; F = 0.04, p = 0.852), nor the interaction between them was significant. The amount of weight-bearing physical activity reported at the worksite was markedly and significantly lower in over 49 years than in the other two age groups (H = 33.15, p < 0.001; Fig. 4 ). Conversely, physical activity at home was higher in women over-49 years than reported in the other two age groups (H = 19.34, p = 0.002; Fig. 5 ). The amount of weight-bearing exercise was low in all three groups, with means about 2 hours per week in the 25- to 35-years, and less than 1 hour per week in the older age groups (Fig. 6 ). About 75% of 25- to 35 years appear to do less than 2.7 hours of weight-bearing exercises per week; while about 75% of women in the older age groups reported doing no exercises (Fig. 6 ). However, the total duration of exercise did not vary significantly among the three age groups, or between women with or without a family history of osteoporosis ( H = 10.25, p = 0.069). Multivariate analysis employing dependent variable of amount of total physical activity and candidate predictors age, educational level, household income, family history of osteoporosis, early menopause and fecundity, appeared to explain only 5% of total variance (R 2 = 0.048, p = 0.038). Use of dependent variable of physical activity at worksite explained about 26% of variance (R 2 = 0.264, p < 0.001; Fig. 7 ); the most effective predictors were educational level (R = 0.361, p < 0.01) and age (R = -0.234, p = 0.002). Similarly, use of dependent variable of physical activity at home explained about 27% of variance (R 2 = 0.273, p < 0.001; Fig. 8 ); again the most effective predictor was age (R = -0.471, p < 0.001). Use of dependent variable of exercise activity explained only 13% of variance (R 2 = 0.128, p < 0.001); the most effective predictor was family history of osteoporosis (R = 0.169, p = 0.011). Total osteoporosis knowledge score was not predicted on the amount of weight-bearing exercise (R = 0.081, p = 0.218). Although, total osteoporosis knowledge scores were significantly associated with WBPA at worksite (R 2 = 0.07, p < 0.001), at home (R 2 = 0.05, p = 0.002) or during exercise (R 2 = 0.04, p = 0.004), squared correlations were rather low in all of cases. Dietary calcium intake Figure 9 summarizes the data on total calcium intake (mg/day) in the three age groups, subdivided into women above and below the poverty line. Independently of income, about 75% of women aged less than 49 years ingest less than 600 mg/day of calcium (i.e. only about 60% of the recommended daily intake, 1000 mg/day). In the over-49 age group (recommended daily intake 1200 mg/day), there is a marked difference in calcium intake between women above and below the poverty line; with women below the poverty line typically showing very low calcium intake (over 75% ingest less than 600 mg/day), whereas calcium intake in women above the poverty line is higher (though nevertheless lower than the recommended daily intake in over 75% of subjects). Calcium intake was estimated through the use of dairy products, with 75% of women ingesting less than 410 mg/day, in all three age groups below the poverty line, and in the two younger age groups above the poverty line. In the over-49 above-poverty-line group, 75% of women ingest less than 775 mg/day. Dairy products contributed on average 57% of total calcium intake in women below the poverty line, versus 68% of total calcium intake in women above the poverty line. Women in the over-49 above-poverty-line group ingested significantly more total calcium (H = 18.36, p = 0.003) and significantly more dairy calcium (H = 18.97, p = 0.002) than women in all other age/income groups (Figs. 10 , 11 ). Supplementary calcium intake did not vary significantly among the age/income groups (H = 9.04, p = 0.108), neither was there any significant interaction between these factors. Multivariate analysis with dependent variable of total calcium intake and candidate predictors age, educational level, household income, lactose intolerance, coffee consumption and use of aluminum antiacids, provided a model that explained only 19% of total variance (R 2 = 0.191, p < 0.001) (Fig. 12 ). With the addition of dairy calcium intake, the model explained 20% of total variance (R 2 = 0.204, p < 0.001) (Fig. 13 ). In both cases, the most effective predictors were lactose intolerance (total calcium intake R = -0.239, p < 0.001; dairy calcium intake R = -0.272, p < 0.001), household income (R = 0.229, p = 0.003; R = 0.232, p = 0.002), age (R = 0.191, p = 0.005; R = 0.198, p = 0.004), and coffee consumption (R = -0.146, p = 0.025; R = -0.141, p = 0.029). Women who had moderate osteoporosis knowledge had an increase in their intake of calcium that was significant (R = 0.142, p = 0.045). Although total osteoporosis knowledge scores were significantly associated with total calcium intake (R 2 = 0.06, p < 0.001) or dairy calcium intake (R 2 = 0.04, p = 0.008) or non-dairy calcium intake (R 2 = 0.03, p = 0.016) or calcium supplements (R 2 = 0.07, p < 0.001), squared correlations were rather low in all cases. With the addition of calcium supplement intake only 12% of total variance was accounted for (R 2 = 0.119, p = 0.003). The greatest predictors were a woman's use of aluminum antiacids (R = 0.232, p < 0.001) and her educational level (R = 0.207, p = 0.004). Discussion The total scores regarding nine knowledge osteoporosis dimensions were similar in all age groups of interviewed women (range of median 14 – 17, average 12.1 – 14.8) out of a possible 42 points. A similar study found Taiwanese women got a mean score of 15 out of 44 points related to six osteoporosis knowledge dimensions [ 21 ], whereas surveyed American women of three age groups got averaged knowledge scores from 32 to 44 points out of 183 [ 11 ]. In all cited cases, the obtained scores indicate that knowledge about osteoporosis is poor or limited among surveyed subjects so health educational programs and health services regarding osteoporosis are necessary for Salvadorean women, as it is also suggested for Taiwanese [ 21 ] and American women [ 11 ] of all ages. The present results also indicate that Salvadorean women with secondary or higher education have significantly better knowledge of osteoporosis than women with a low educational level, regardless of age. Similarly, other study found that better educated Chinese women in Singapore seem to know more about osteoporosis than those ones worst educated [ 22 ]. A previous study has likewise found that osteoporosis-related knowledge is independent of age [ 11 ]. Our multivariate analyses indicated that the most effective predictors of osteoporosis-related knowledge were educational level, household income, and early menopause. This latter factor perhaps affects osteoporosis-related knowledge through to give brochures and magazines to women at shopping centers, supermarkets, physician's clinics, schools, and colleges by non governmental organizations such as Salvadorean Demographical Association, dairy good producers such as New Zealand Dairy Board and Australian Milk Products, and some pharmaceutical laboratories, especially those produce calcium supplements. As well as information presented on television and in the press. Besides, short counselling sessions about preventive aspects of osteoporosis are given by some physician's private clinics, because there is not a settled public health education program about osteoporosis in El Salvador. This country is not only case in Latin America, since Mexican women got more osteoporosis information from mass communication media than health education activities of public institutions [ 14 ]. However, total knowledge about osteoporosis may not lead to an improvement in health lifestyle; it is necessary to know more about some specific aspects as osteoporosis risk factors and to acquire healthy habits to reduce the risk for low bone mass. In multivariate analyses, knowledge about osteoporosis was not a significant predictor of either amount of physical activity or total calcium intake. Previous studies likewise found that knowledge of osteoporosis does not correlate with risk-reducing life habits [ 11 , 12 ]. These authors suggested that osteoporosis-related knowledge among women of their sample was limited, the information was not fully understood or poorly internalized. They also suggested that it is unlikely that this type of knowledge will provide a basis for decisions about life style or unhealthy habits [ 11 ]. These comments are perhaps similarly applicable to the subjects in our study. Other previous studies have obtained results consistent with this view that osteoporosis-related knowledge is often poorly integrated and internalized, and does not lead to improved health behaviours [ 12 , 16 , 23 , 24 ]. Our data on physical activity in the three age groups indicate that the amount of physical activity engaged in paid work currently declines with increasing age, whereas the amount of physical activity during housework appears to increase with increasing age. This trend was confirmed by multivariate analysis, which indicates that the most effective predictors of amount of physical activity at worksite or at home were age and educational level. Educational level attained likely increases the likelihood of obtaining paid work. However, recreational activities and other health-preventive behaviours such as to do isometric exercises may also determine total physical activity. Considering all subjects together, most reported physical activity (96%) was performed in the course of daily housework, walking to work or shops, or standing at work or at home; only 4% was directly applicable to weight-bearing exercise. About 50% of women in the youngest age group did more than 2 hours of physical activity per week, but the amount of physical activity in the older women was markedly lower, and even in the youngest age group very few women performed the recommended daily minimum of 30 minutes of exercise per day [ 5 , 6 ]. Similar results were obtained in a previous study, which found that fewer than 50% of African-American and Hispanic women interviewed in the US do 60 minutes of physical exercise per week [ 16 ]. Very few of the women interviewed for the present study employ any of the weight-bearing physical activities that are known to be especially effective for increasing bone mass and thus perhaps reducing the risk of osteoporosis, such as isometrics [ 25 ]. Similar findings have been obtained in studies of Caucasian women in the US [ 11 , 12 ] and of African-American and Hispanic women residing in the US [ 12 , 16 ]. Independent of age and income, most women interviewed (50 – 75%) have a daily calcium intake of less than 60% of the recommended level, thus increasing the risk of osteoporosis. Similarly, in another studies of Caucasian and African-American and Hispanic women in the US [ 12 , 16 ], and Asian and Caucasian women in Australia [ 13 ], most of these women did not fulfil the suggested calcium intake. By contrast, in other recent study of Caucasian women in the US [ 11 ], only about 20% of women ingested less than 60% of the recommended daily calcium. The difference between our results in El Salvador and those obtained in the study of Caucasian women in the US [ 11 ] may, at least in part, reflect differences in standard of living between these two countries. Specifically, the generally higher incomes in the US may be associated with healthier diet, higher educational level, better access to healthcare, and better public education about general health and the prevention of diseases like osteoporosis. Additionally, calcium fortified foods are more readily available in the US. The average proportion of calcium intake as dairy products was 57% in women below the poverty line and 68% in women above the poverty line; both values are close to those reported for Caucasian women in the US [ 11 ]. These authors did not detect any significant variation with income, but note that their sample was smaller (n = 75) and more homogeneous (in terms of educational level and household income) than the sample of our study. The relationships between household income and calcium intake observed in our multivariate analyses likewise indicate that calcium intake varies with income. The other important predictor of calcium intake in this analysis was lactose intolerance, reported by 19% of the women interviewed; dairy calcium intake was likely lower in these women. As lightly lower prevalence of lactose intolerance (16%) was reported in the study of Caucasian women in the US [ 11 ]. The consumption of caffeine-containing drinks, especially coffee, has been shown to influence calcium intake, since these beverages often replace milk and milk-based beverages due to dairy products are relatively expensive in El Salvador and many Salvadorean families (42.9%) have a low purchasing power, and live below the poverty line [ 26 ]. Excessive caffeine ingestion is also reported to accelerate bone resorption [ 5 , 6 ], and prevents intestinal calcium absorption [ 9 ]. Calcium intake also varied with age, being significantly less among younger women. Similarly, most of interviewed Caucasian and African-American young adult women in the US did not get recommended calcium intake per day [ 12 ]. This may be because younger women tend to perceive dairy products as having a high content in animal fats, and thus tend to reject them, as it has been suggested by previous authors [ 19 ]. Auld et al . also suggested that Hispanic women (particularly adolescents and young women) in general tend to reject dairy products more than Anglosaxon and Asiatic women. The key practical implications of this study relate to suggest set up of primary healthcare programs regarding osteoporosis for Salvadorean women as it has also suggested for Taiwanese women [ 21 ], and Hispanic and African-American women [ 16 ], as well as training of healthcare professionals, which in light of the present study need to pay special attention to the following aspects of osteoporosis prevention: a) The type and frequency of physical exercise. b) Diet-related risk factors, including inadequate intake of calcium, vitamin D, and phosphorus; also the adverse effects of drinking coffee or other low-calcium drinks instead of milk or milk-based drinks. c) The importance of monitoring menstruation frequency, since normal circulating estrogen levels and normal menstrual cycles are important for maintaining normal bone metabolism. In addition, it is clearly important to set up health education programs to focus on integration and internalization of knowledge about osteoporosis. Although our results indicate that many women have modest knowledge about osteoporosis concerning risk factors and preventive behaviours, this knowledge often does not translate to appropriate changes in healthy life habits as it is shown through a weak association between total osteoporosis scores and exercise or calcium intake, so that osteoporosis knowledge is not well internalized among interviewed Salvadorean women. The lack of time to practice most adequate osteoporosis preventive exercise may accounts for the weak association between foregoing variables since majority of interviewed women perform WBPA (> 8 hours/day), but as labour activities or housework. The low calcium intake among subjects may be explained through they have a low purchasing power and do not have the habit to ingest enough dairy products, the main diet calcium source [ 9 ]. Non-dairy calcium intake including supplements was also low (< 370 mg/day), so that they did not fulfil the recommended daily calcium intake from this source either. Finally, we would suggest that osteoporosis education campaigns should be directed at families as well as individuals, with the aim of fomenting within-family education of children about the importance of calcium in the diet, whether as dairy products or in other calcium-rich products such soya milk and oat-based drinks and desserts. Conclusions It showed that Salvadorean women with secondary or higher education have significantly better knowledge about osteoporosis than women with lower educational level, regardless of age. However, this knowledge does not appear to lead to improved life style or preventive habits of osteoporosis. The amount of weight bearing physical activity (mainly walking or standing) during paid work or housework was high in the three age groups of interviewed women (over 8 hours/day). However, very few of the women interviewed for this study did not perform any of the weight-bearing physical activities that are known to be especially effective for increasing bone mass and thus reducing the risk of osteoporosis, such as going up and down stairs, jogging, aerobics, swimming, and isometrics. Most Salvadorean interviewed women have a daily calcium intake of 60% or less than the recommended level depending of lactose intolerance, household income, age, and coffee consumption. The former variables directly affect both calcium source diversity and intake frequency. Age would influence dairy calcium intake, since younger women tend to perceive dairy products as having a high animal fat content, and thus reject them. The coffee consumption would affect calcium intake, since this beverage often replace milk. Therefore, all these factors together may be increasing the risk to develop osteoporosis among Salvadorean women. List of abbreviations WBPA: Weight-bearing physical activity ANOVA: Analysis of Variance SEM: Standard error of mean Competing interests None declared. Authors' contributions RHR performed the design of the study, coordinated the study, performed data analysis, and drafted the paper. SMG participed in the design of the study and provided input into the paper. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC517502.xml
550675	Dispersion of cardiac action potential duration and the initiation of re-entry: A computational study	Background The initiation of re-entrant cardiac arrhythmias is associated with increased dispersion of repolarisation, but the details are difficult to investigate either experimentally or clinically. We used a computational model of cardiac tissue to study systematically the association between action potential duration (APD) dispersion and susceptibility to re-entry. Methods We simulated a 60 × 60 mm 2 D sheet of cardiac ventricular tissue using the Luo-Rudy phase 1 model, with maximal conductance of the K + channel gKmax set to 0.004 mS mm -2 . Within the central 40 × 40 mm region we introduced square regions with prolonged APD by reducing gKmax to between 0.001 and 0.003 mS mm -2 . We varied (i) the spatial scale of these regions, (ii) the magnitude of gKmax in these regions, and (iii) cell-to-cell coupling. Results Changing spatial scale from 5 to 20 mm increased APD dispersion from 49 to 102 ms, and the susceptible window from 31 to 86 ms. Decreasing gKmax in regions with prolonged APD from 0.003 to 0.001 mS mm -2 increased APD dispersion from 22 to 70 ms, and the susceptible window from <1 to 56 ms. Decreasing cell-to-cell coupling by changing the diffusion coefficient from 0.2 to 0.05 mm 2 ms -1 increased APD dispersion from 57 to 88 ms, and increased the susceptible window from 41 to 74 ms. Conclusion We found a close association between increased APD dispersion and susceptibility to re-entrant arrhythmias, when APD dispersion is increased by larger spatial scale of heterogeneity, greater electrophysiological heterogeneity, and weaker cell-to-cell coupling.	1. Background Cardiac disease remains an important cause of sudden death in the industrialised world, and in many cases the lethal events are the cardiac arrhythmias called ventricular tachycardia (VT) and ventricular fibrillation (VF). Spontaneous episodes of VT and VF occur in patients where cardiac disease or congenital abnormality has remodelled either the structure or function of cardiac cells and tissue. There is abundant experimental evidence to support the idea that VT and VF are sustained by re-entry [ 1 , 2 ], but the initiation of re-entry in a particular individual is not well understood, and so is difficult to either predict or prevent. Slow conduction and unidirectional block have long been known to facilitate re-entry [ 3 ], and experimental studies have established a link between regional differences in repolarisation, and an increased vulnerability to re-entrant arrhythmias following one or more premature stimuli [ 4 - 7 ]. One of the earliest computer models of activation in cardiac tissue was used to demonstrate that regional differences in repolarisation can allow fibrillation to develop following a premature stimulus [ 8 ]. Further experimental studies have found that steep gradients in repolarisation correlate with arcs of conduction block around which re-entry circulates [ 9 - 12 ], and have suggested that regions with longer refractory period must be of a critical size for sustained re-entry to occur [ 13 ]. Computational and theoretical studies [ 14 - 16 ] have also shown how a region with prolonged repolarisation can block a premature excitation resulting in initiation of re-entry, and that the size of the inhomogeneity determines the characteristics and persistence of re-entry. Regional differences in repolarisation are often described as action potential duration (APD) dispersion. The difference between the longest and shortest observed APD is a conceptually simple and easily obtained quantity and has widely been used to measure APD dispersion, although other indices have been proposed [ 17 ]. Some experimental studies have established critical values of APD dispersion above which re-entry is initiated consistently [ 18 ]. In others the gradient of APD has been measured, and spatial gradients of between 2 and 12.5 ms mm -1 were associated with block and re-entry [ 9 , 19 , 20 ]. Spatial APD gradients arise from regional differences in ion channel function, but their magnitude depends on electrotonic current flow during repolarisation. APD dispersion can be produced by the spatial scale of regional differences and the magnitude of functional heterogeneity, and is modulated by electrotonic current flow which depends on the strength of cell-to-cell coupling [ 16 , 21 , 22 ]. The relative effect of these three quantities on APD dispersion and vulnerability to re-entry is important because both disease and congenital abnormalities can result in changes to one or more of them. However, it is difficult to control these tissue properties independently in experiments. Computational models offer a powerful research tool for addressing these questions, because the properties of a virtual tissue can be controlled precisely and independently in a way that would be extremely difficult to achieve experimentally. The purpose of this study was therefore to investigate systematically how measured APD dispersion and vulnerability to re-entry in a computational model of ventricular tissue are related to: (i) the spatial scale of heterogeneity, (ii) the magnitude of differences in K + channel conductance between regions with short and long APD, and (iii) strength of cell-to-cell coupling. 2. Methods 2.1 Computational model of electrical activation We simulated electrical activation in a 2 D isotropic monodomain virtual tissue [ 23 ] where V m is membrane voltage, C m specific membrane capacitance, D a diffusion coefficient and I ion current flow through the cell membrane per unit area. We used the Luo-Rudy phase 1 (LR1) model [ 24 ] to give I ion , where I Na , I Ca (described as I si in the original model) and I K are time and voltage dependent currents flowing through Na + , Ca 2+ , and K + channels, I K 1 a time-independent K + current, I Kp a plateau K + current, and I b a background current. We changed two parameters from the original Luo and Rudy paper [ 24 ]. We reduced maximum Na + conductance from 0.23 mS mm -2 to 0.16 mS mm -2 as in the later version of the model [ 25 ], and we reduced the maximum conductance of the slow inward current from 0.0009 mS mm -2 to 0.0005 mS mm -2 to produce an APD comparable to that in the canine ventricle. We controlled repolarisation by varying maximum K + conductance ( gKmax ) from the default value of 0.00282 mS mm -2 to a value between 0.001 mS mm -2 and 0.004 mS mm -2 (see below). 2.2 Numerical methods We solved equation 1 and the LR1 equations using an explicit Euler method, with both a lookup table of the voltage dependent parameters in the LR1 model, and an adaptive operator splitting technique [ 26 ]. We applied no-flux boundary conditions, set C m to 0.001 μF mm -2 , and set D to between 0.05 and 0.2 mm 2 ms -1 . We used an adaptive timestep of either 0.02 or 0.1 ms depending on the magnitude of dV m / dt at each grid point [ 26 ]. With a space step of 0.2 mm, and D of 0.1 mm 2 ms -1 we obtained a conduction velocity (CV) for a stable plane wave of 0.56 m s -1 , with a speedup of about two times and an error in CV of 2.5 % compared to computations with a fixed timestep of 0.01 ms. Simulations with smaller fixed and adaptive timesteps yielded plane waves with a comparable CV. Changing the space step to 0.25 mm and 0.15 mm resulted in a change of CV for a plane wave of <5 % compared to the CV computed with a space step of 0.2 mm. These findings indicated the stability of our numerical method. Figure 1 shows action potentials, APD restitution curves, and CV restitution for virtual tissue with different values of gKmax . In each case a propagating action potential could be elicited with a minimum diastolic interval of about 10 ms, indicating that the refractory period was very close to the APD. Figure 1 (a) Action potentials recorded during steady pacing at 500 ms intervals for different values of gKmax . (b) Action potential duration restitution and (c) conduction velocity restitution for different values of diastolic interval measured with a premature stimulus during steady pacing at 500 ms intervals. All measurements obtained from a narrow strip of virtual tissue 10 mm long and with uniform gKmax of 0.001, 0.002, 0.003, and 0.004 mS mm -2 as indicated. 2.3 Virtual tissue and heterogeneity Each of the virtual tissues in this study represented a 60 × 60 mm 2 D sheet with a 10 mm border around each edge with gKmax set to 0.004 mS mm -2 . The central 40 × 40 mm region was heterogeneous, and was subdivided into squares. Within alternate squares gKmax was set to either 0.004 mS mm -2 or to a specific lower value (see below). Thus the virtual tissues had a border region with short APD, and a central heterogeneous region divided into a chequerboard with alternating squares of either short or long APD (Figure 2 ). Figure 2 Configuration of 60 × 60 mm virtual tissues used in the study where (a) spatial scale, (b) Δ gKmax , and (c) strength of cell-to-cell coupling were varied as indicated on the figure. Greyscale shows gKmax , in each region, with light grey, mid grey, dark gray and black corresponding to gKmax values of 0.004, 0.003, 0.002, and 0.001 mS mm -2 respectively. In our reference virtual tissue, the 40 × 40 mm heterogeneous region was divided into 16 squares giving heterogeneity with a spatial scale of 10 mm. In half of the 16 squares gKmax was set to 0.001 mS mm -2 , giving a functional heterogeneity with a difference in gKmax (Δ gkmax ) of 0.003 mS mm -2 . The diffusion coefficient in the whole virtual tissue was set to 0.1 mm 2 ms -1 . We varied the spatial scale of heterogeneity by changing the size of heterogeneity in the reference virtual tissue from 10 mm to 20 mm and 5 mm (Figure 2a ), varied Δ gkmax from 0.003 to 0.002 and 0.001 mS mm -2 by changing gKmax in the alternate squares from 0.001 to 0.002 and 0.003 mS mm -2 respectively (Figure 2b ), and varied the strength of cell-to-cell coupling by changing the diffusion coefficient from 0.1 to 0.05 and 0.2 mm 2 ms -1 (Figure 2c ). 2.4 APD dispersion Action potentials were initiated in each virtual tissue by holding the membrane voltage along one edge at 0 mV for 2 ms. We measured APD to 90% recovery (APD 90 ) at every grid point. We estimated APD dispersion during steady pacing with three measured that have been used in experimental studies [ 17 ]. First we measured APD across the whole virtual tissue, and determined the difference between the maximum and minimum APD ( APDdiff ). Second we measured the standard deviation of APD ( APDSD ) across the whole virtual tissue. Finally we measured the APD difference between each grid point and its neighbours 1 mm above, below, left and right, and determined the maximum value of measurements throughout the whole virtual tissue ( maxLD ) [ 17 ]. 2.5 Vulnerability to re-entry A spatially homogenous control virtual tissue with uniform gKmax supported propagating plane waves following S1 stimulation along one edge. These propagating plane waves had a depolarising wavefront and a repolarising wave back both aligned parallel to the edge that was stimulated. A premature S2 stimulus delivered to the same edge as the S1 stimulus therefore resulted either in block or in a propagating plane wave. In the heterogeneous virtual tissues the wave back was not a plane wave because some regions repolarised more quickly than others. A premature S2 stimulus could produce a wavefront that would encounter a mixture of recovered and refractory tissue, and hence elicit wavebreak and re-entry. We therefore assessed vulnerability to re-entry in the heterogeneous virtual tissues by delivering two S1 stimuli to one edge at 500 ms intervals, and then a premature S2 stimulus to the same edge. We varied the timing of the S2 stimulus in steps of 1 ms. The virtual tissue response was characterised as either block if the S2 stimulus failed to propagate, re-entry if the S2 stimulus elicited re-entry that completed more than one cycle, wavebreak if the S2 stimulus elicited a wave that broke but did not re-enter, or propagation if the S2 stimulus elicited a wave that propagated without wavebreak. Vulnerability to re-entry is typically estimated by applying a local premature stimulus that interacts with repolarising tissue, and the vulnerable window is the range of stimulus strength and timing that elicits re-entry. In this study, we investigated the initiation of re-entry by S1 S2 stimulation from the same stimulus site, and we estimated the vulnerability of each virtual tissue to re-entry from the range of S2 intervals that resulted in either wavebreak or re-entry. To avoid confusion we refer to our estimate of vulnerability as susceptibility to re-entry, and to the range of S2 intervals as the width of the susceptible window. 2.6 Potential antiarrhythmic strategies We made a preliminary investigation into two candidate mechanisms for reducing susceptibility, inactivation of the Na + channel and conductance of the time independent K + channel g K 1 . Block of an action potential occurs if there is insufficient Na + current to support a propagating wavefront [ 27 ]; when Na + channels have not recovered from inactivation, then a propagating wave blocks and dissipates [ 28 ]. Na + channel inactivation is controlled by the j -gate in the LR1 model [ 24 ]. We prolonged recovery of Na + channels from inactivation throughout the virtual tissue by multiplying the time constant of Na + channel inactivation τ j by 10 [ 29 ]. The time independent K + current i K 1 is a voltage dependent current that holds the membrane at its resting potential. It is activated during repolarisation and at rest, and is also activated close to the core of re-entrant waves [ 30 , 31 ]. We investigated the effect of doubling the conductance of i K 1 throughout the virtual tissue. 3. Results 3.1 Propagation and APD dispersion during pacing Figure 3 shows the spatial distribution of APD 90 in variants of the virtual tissue during pacing at a cycle length of 500 ms from the bottom edge. The three columns show the effect of changing spatial scales (Figure 3a ), Δ gkmax (Figure 3b ), and strength of cell-to-cell coupling (Figure 3c ). The range of colours and number of contours on each figure indicates the range of APD. Hence the leftmost column shows that when Δ gkmax and the strength of cell-to-cell coupling are held constant, increasing spatial scale increases the range of APD and decreasing spatial scale decreases the range of APD. Overall, Figure 3 shows that small spatial scales, small Δ gkmax , and strong cell-to-cell coupling act to reduce the range of observed APD. This effect can also be seen in Figure 4 , which summarises how the three measures of APD dispersion were affected by spatial scale, Δ gkmax , and strength of cell-to-cell coupling. Each of these measures changed monotonically within the range of spatial scale, Δ gkmax and strength of cell-to-cell coupling that we studied. APDdiff was halved by reducing spatial scale from 20 to 5 mm or by increasing the diffusion coefficient from 0.05 to 0.1 mm 2 ms -1 . Figure 3 Spatial distribution of APD during from pacing at 500 ms intervals in each variant of the 60 × 60 mm virtual tissue. (a) Effect of changing spatial scale, with Δ gKmax fixed at 0.003 mS mm -2 , and diffusion coefficient fixed at 0.1 mm 2 ms -1 . (b) Effect of changing Δ gKmax , with spatial scale fixed at 10 mm, and diffusion coefficient fixed at 0.1 mm 2 ms -1 . (c) Effect of changing strength of cell-to-cell coupling by changing the diffusion coefficient, with spatial scale fixed at 10 mm, and Δ gKmax fixed at 0.003 mS mm -2 . Figure 4 APDdiff , APDSD , and MaxLD measured in virtual tissues where (a) the spatial scale of heterogeneity, (b) magnitude of functional heterogeneity, and (c) strength of cell-to-cell coupling were changed. When re-entry was initiated, we observed break-up into multiple re-entrant wavelets with up to 18 phase singularities. The mechanism of instability was likely to be a combination of the spatial heterogeneity leading to localised conduction block combined with dynamical instability resulting from steep APD restitution [ 32 ] (Figure 1 ), but was not investigated explicitly. In some simulations the re-entrant waves coalesced and re-entry spontaneously terminated. However, there was no clear association between this observation and S2 timing, spatial scale, functional heterogeneity, or coupling. 3.2 Susceptibility to re-entry Figure 5 shows examples of re-entry, wavebreak and propagation in the reference virtual tissue. In each case the pacing (S1), and premature (S2) stimuli were delivered to the bottom edge. In the top row (Figure 5a – see also the movie in additional file 1 ), the premature S2 activation was blocked at each of the regions with prolonged repolarisation, and curled round to give figure-of-8 re-entry. In the second row (Figure 5b – see also the movie in additional file 2 ) the S2 stimulus was 15 ms later, and although the S2 activation was partially blocked, re-entry was prevented by collision of the wavebreak with antegrade activation of the regions with prolonged APD. With a later S2 stimulus (Figure 5c – see also the movie in additional file 3 ), the regions with prolonged APD had recovered enough to conduct the S2 activation, although the activation wave was delayed slightly by each region with prolonged APD. Figure 5 Example responses to premature S2 stimulus in virtual tissue with heterogeneity on spatial scale of 10 mm, Δ gKmax of 0.003 mS mm -2 , and diffusion coefficient set to 0.1 mm 2 ms -1 . Blue lines outline regions with prolonged repolarisation.. (a) S2 at 230 ms and induction of re-entry. (b) S2 at 250 ms, wavebreaks are indicated with a star. (c) S2 at 265 ms and delayed propagation with no wavebreak. In (a-c) snapshots 50, 100 and 150 ms after the S2 stimulus are included, with propagation from bottom to top of the figure. Isochrones at intervals of 5 ms are shown, and colour coding indicates the progress of the wavefront. Movies of the simulations shown in this figure are available as additional files Figure 5a Figure 5b, and Figure 5c. Figure 6 shows the detailed response of each virtual tissue for a range of S2 intervals, and indicates how each of the three interventions affects the response of the tissue to a premature stimulus. Increasing spatial scale, increasing Δ gkmax , and decreasing the strength of cell to cell coupling all resulted in an greater range of S2 intervals that resulted in wavebreak (red) or re-entry (orange), and hence an increase in the width of the susceptible window The lower bound of the susceptible window where S2 was blocked depended on the refractory period of the border tissue with short APD, and was not greatly affected by changes in spatial scale, Δ gkmax or strength of cell-to-cell coupling. The upper bound showed a similar trend to the measures of APD dispersion shown in Figure 4 , with a large spatial scale, large Δ gkmax and weak cell-to-cell coupling associated with a wide susceptible window. Figure 6 Response of virtual tissues to premature S2 stimulus. Blue indicates block, orange re-entry, red wavebreak, and purple propagation. (a) Results for changing in spatial scale, (b) changes in Δ gKmax , and (c) changes in strength of cell-to-cell coupling, where D is the diffusion coefficient. The virtual tissue with a spatial scale of 20 mm showed a different pattern of susceptibility compared to the others, with two ranges of S2 that initiated wavebreak and two ranges of S2 that initiated re-entry. This behaviour is illustrated in Figure 7 . For S2 delivered between 198 and 226 ms, the regions with prolonged APD blocked the premature activation, and re-entry was initiated by retrograde activation through the isthmus between these two regions (Figure 7a – see also the movie in additional file 4 ). For values of S2 between 227 and 249 ms, the isthmus conducted the S2 activation resulting in wavebreak, but the regions with prolonged APD remained refractory (Figure 7b – see also the movie in additional file 5 ). For values of S2 between 250 and 263 ms, re-entry was initiated by retrograde activation of the regions with prolonged APD (Figure 7c – see also the movie in additional file 6 ). Figure 7 (See text for details) Example responses to premature S2 stimulus in virtual tissue with heterogeneity on spatial scale of 20 mm and Δ gKmax of 0.003 mS mm -2 . Arrows show direction of propagation. (a) S2 at 200 ms and induction of re-entry with two phase singularities. (b) S2 at 245 ms and broken wave, retrograde activation is blocked. (c) S2 at 255 ms with retrograde activation and re-entry with four phase singularities i.e. two systems of figure-of-eight re-entry In each figure isochrones at intervals of 5 ms are shown, and the activation wavefront at S2+150 ms (a and c) and S2+155 ms (b) is shown as a thick black line. Movies of the simulations shown in this figure are available as additional files Figure 7a, Figure 7b, and Figure 7c. Figure 8a shows the width of the susceptible window plotted against APDdiff and reveals an approximately linear relationship. The correlation coefficient R 2 = 0.99, which indicates a strong association between APDdiff and susceptibility. The interception of the line with the APDdiff axis also suggests that for the S1 S2 configuration used in this study, the susceptible window falls to zero for APD dispersion less than 20 ms. The association between the other measures of APD dispersion ( APDSD figure 8b , maxLD Figure 8c ) and susceptibility is also monotonic, but less well correlated than for APDdiff . Figure 8 Association between width of the susceptible window and (a) APDdiff , (b) APDSD , and (c) MaxLD , for each of the three interventions In each case the grey circle indicates the reference virtual tissue with spatial scale of 10 mm, Δ gKmax of 0.003 mS mm -2 and diffusion coefficient set to of 0.1 mm 2 ms -1 . 3.3 Potential antiarrhythmic strategies Although prolonging τ j by a factor of 10 had only a small (< 2 ms) effect on maximum and minimum APD, the width of the susceptible window was decreased from 56 ms to 39 ms. The lower bound of the susceptible window moved from 198 to 216 ms, reflecting an increase in the refractory period of the virtual tissue as well as more prominent conduction velocity restitution. Doubling g K 1 increased current flow across the membrane during repolarisation, shortening APD by about 10% and decreasing APDdiff from 70 ms to 49 ms. The width of the susceptible window was also reduced from 56 ms to 42 ms when g K 1 was doubled. The lower bound of the susceptible window moved from 198 ms to 175 ms, as a result of the shorter APD. 4. Discussion In this study we have used a computational model of cardiac tissue to dissect out the effects of spatial scale, Δ gkmax , and strength of cell-to-cell coupling on APD dispersion and susceptibility to re-entry. Wavebreaks and re-entry can be created in cardiac tissue when an activation wavefront encounters a gradient of recovery. Experimental studies have therefore found an association between increased APD dispersion and greater susceptibility to re-entry because a premature stimulus is more likely to be blocked in tissue with regions of prolonged APD. In this study we found that large spatial scale heterogeneity, large Δ gkmax , and reduced strength of cell-to-cell coupling all increased both APD dispersion and susceptibility to re-entry. Tissue heterogeneities produce APD dispersion, and APD dispersion is modulated by electrotonic current flow [ 21 ]. In this study, spatial scale and Δ gkmax affected APD dispersion directly, whereas changing the strength of cell-to-cell coupling affected electrotonic current flow. This study indicates that each of these factors could be an important component in arrhythmogenesis, and that the susceptibility of a heterogenous tissue to re-entry can be estimated from simple measures of APD dispersion. 4.1 Relation to other work Although the spatial scale of heterogeneity has been identified as potentially important in other studies [ 13 ], there is little information in the experimental literature to indicate how spatial scale affects susceptibility to re-entry. In one experimental study a ~1 cm2 region of thin layer of rabbit ventricular epicardium was cooled to produce a small region with prolonged APD, producing a dispersion of refractory periods ranging from 27 and 45 ms [ 10 ]. Re-entry could be initiated in this preparation using 4 increasingly premature stimuli. The spatial scale of heterogeneity in this experimental study was comparable to the reference virtual tissue used in our present study, but the effects of Δ gkmax and strength of cell-to-cell coupling in the experimental study are difficult to establish. Nevertheless the initiation of re-entry by block and retrograde activation in the region with prolonged APD followed a broadly similar pattern to our simulations, although the activation pathways in the experimental study were more complex, presumably due to anisotropic conduction in the rabbit ventricle. A decrease in strength of cell-to-cell coupling results in slowed conduction, and this is a common finding in tissue damaged by ischaemia, infarction [ 33 ], and other pathology [ 34 ]. Several studies have shown that decreasing cell-to-cell coupling can expose ionic heterogeneities [ 22 , 35 - 37 ]. Recent computational studies have addressed the influence of heterogeneous acetylcholine distribution on the vulnerability to and stability of re-entry in the atria [ 15 , 32 ]. The findings of these studies are broadly similar to the present study, although the effects of cell-to-cell coupling were not explicitly addressed, and initiation of re-entry was by either crossfield stimulation [ 32 ] or by S1 and S2 stimulation at different sites [ 15 ]. Both of these protocols would be expected to induce re-entry even in uniform tissue. In the present study both S1 and S2 stimuli were delivered from the same location, which does not initiate re-entry in uniform tissue, allowing us to examine the effect of heterogeneity on the initiation of re-entry in isolation. The dynamical behaviour of APD is recognised as important not only for the stability of re-entry [ 2 ] but also in the development of alternans [ 38 ]. Recent experimental [ 39 ] and computational [ 38 , 40 ] studies have shown that APD dispersion can arise dynamically leading to discordant alternans, wavebreak, and re-entry in tissue that is either homogeneous or in which the ionic properties vary smoothly[ 38 , 41 , 42 ]. In the present study we measured APD dispersion at a fixed cycle length of 500 ms. The APD restitution curves given in Figure 1 indicate that APD dispersion could have been affected by pacing at shorter cycle lengths. This observation raises the possibility that heterogenous APD restitution could act to amplify APD dispersion. The effect of heterogeneity on the stability of spiral waves has been investigated by Xie et al [ 43 ]. This study found that the amount of heterogeneity required to destabilise re-entry decreased as the degree of dynamical instability resulting from a steep APD restitution curve increased. In the present study we were interested in the initiation of re-entry rather than the stability of re-entry once initiated. 4.2 APD dispersion and susceptibility to arrhythmias Normal ventricular tissue is remarkably resistant to the initiation of re-entry, but this robustness is greatly reduced by actions that increase the spatial dispersion of refractoriness. In this computational study we have shown that regional differences in repolarisation have an interlinked effect not only on the initiation of re-entry but also on measures of APD dispersion. Measures of APD dispersion are valuable in clinical practice because they could provide an estimate of arrhythmia risk, and various indices have been developed in experimental studies [ 17 ]. In our present study we have found that relatively simple measures of APD dispersion obtained from the tissue were related to the width of the susceptible period for re-entry. 4.3 Potential antiarrhythmic strategies These preliminary investigations suggest that, in our model, susceptibility to re-entry could be reduced if recovery of Na + channels from inactivation can be prolonged, or if the conductance of the i K 1 channel can be increased. The effect of this kind of intervention in the intact heart may however be more complex. Other computational studies have shown that modifying the kinetics of the Na + channel can have a pro-arrhythmic effect. Delaying recovery of Na + channels from inactivation can increase the slope of the APD restitution curve and hence the likelihood of alternans and re-entry [ 29 ], and reducing Na + channel conductance increases the vulnerable window [ 44 ]. Differences in the spatio-temporal complexity of VF between left and right ventricles have been attributed to differences in the current density of the i K 1 channel in experimental studies [ 30 ]. Although this experimental finding is not directly connected to the effects of the i K 1 channel conductance on susceptibility to re-entry investigated in the present study, it does highlight the potential importance of this channel for the mechanisms of re-entry. The influence of individual ion channel currents on the initiation and subsequent behaviour of re-entry is an important direction for future research, but will require more biophysically detailed cell models than the LR1 model used in this study. 4.4 Limitations of the study The electrical behaviour of cardiac tissue is complex, and depends on processes that act at tissue, cell, sub-cellular, and molecular levels. Computational models of electrical activation and conduction in the heart aim to simulate processes that are relevant to the research question, and simplifications are made accordingly. This study involved a large number of computations to establish susceptibility to re-entry, and so we chose to use a model that was a compromise between fidelity to real cardiac tissue and computational requirements. More detailed versions of the LR model and others incorporating a fuller description of ion channels, pumps, exchangers, as well as Ca 2+ storage and release have been developed [ 22 , 25 , 45 , 46 ]. In tissue with regional ischaemia, Ca 2+ handling may become heterogeneous in addition to APD, and so it is possible that susceptibility to re-entry could also be modified if this additional feature is taken into account. In the present study we chose to use an idealised geometrical heterogeneity based on square regions because this approach allowed us to assess the initiation of re-entry under well controlled conditions. In real cardiac tissue we would expect the heterogeneities to be much more irregular in shape and gradient, and the conditions that favour re-entry to be dependent on the relative location of the heterogenous region and the stimulus site. The behaviour of re-entry in 3 D tissue is more complex than in 2 D, especially when the effects of rotational anisotropy and transmural differences in action potential shape and duration are taken into account [ 47 ]. Studies relating APD dispersion and susceptibility to re-entry in anatomically detailed 3 D tissue are another important project for the future. Recent computational studies indicate that the mechanical properties can not only modify the behaviour of re-entrant waves [ 48 ], but also that stretch activated channels in the cell membrane can contribute to susceptibility to re-entry if the tissue is stretched during repolarisation [ 49 , 50 ]. Since electrical repolarisation occurs at the same time as force generation in cardiac cells, the effect of cardiac mechanics on susceptibility to re-entry remains an important research question. Authors' contributions RHC conceived and designed the study, wrote the simulation code, and ran the simulations. AVH participated in the study design, and helped draft the manuscript. Both authors read and approved the final manuscript. Supplementary Material Additional File 1 Movie relating to Figure 5a Click here for file Additional File 2 Movie relating to Figure 5b Click here for file Additional File 3 Movie relating to Figure 5c Click here for file Additional File 4 Movie relating to Figure 7a Click here for file Additional File 5 Movie relating to Figure 7b Click here for file Additional File 6 Movie relating to Figure 7c Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC550675.xml
550661	Sea urchin vault structure, composition, and differential localization during development	Background Vaults are intriguing ribonucleoprotein assemblies with an unknown function that are conserved among higher eukaryotes. The Pacific coast sea urchin, Strongylocentrotus purpuratus , is an invertebrate model organism that is evolutionarily closer to humans than Drosophila and C. elegans , neither of which possesses vaults. Here we compare the structures of sea urchin and mammalian vaults and analyze the subcellular distribution of vaults during sea urchin embryogenesis. Results The sequence of the sea urchin major vault protein (MVP) was assembled from expressed sequence tags and genome traces, and the predicted protein was found to have 64% identity and 81% similarity to rat MVP. Sea urchin MVP includes seven ~50 residue repeats in the N-terminal half of the protein and a predicted coiled coil domain in the C-terminus, as does rat MVP. A cryoelectron microscopy (cryoEM) reconstruction of isolated sea urchin vaults reveals the assembly to have a barrel-shaped external structure that is nearly identical to the rat vault structure. Analysis of the molecular composition of the sea urchin vault indicates that it contains components that may be homologs of the mammalian vault RNA component (vRNA) and protein components (VPARP and TEP1). The sea urchin vault appears to have additional protein components in the molecular weight range of 14–55 kDa that might correspond to molecular contents. Confocal experiments indicate a dramatic relocalization of MVP from the cytoplasm to the nucleus during sea urchin embryogenesis. Conclusions These results are suggestive of a role for the vault in delivering macromolecules to the nucleus during development.	Background The sea urchin Strongylocentrotus purpuratus is an important model system in developmental biology and its genome is currently being sequenced by the Human Genome Sequencing Center at the Baylor College of Medicine under the auspices of the National Human Genome Research Institute (NHGRI). Sea urchin embryos are well suited for biochemical approaches to studying the cell biology of development, as large quantities of eggs can easily be obtained, and their fertilization initiates the synchronous development of optically transparent embryos [ 1 ]. Sea urchins occupy an important phylogenetic position as basal deuterostomes, and are thus more closely related to humans than are other invertebrate model organisms such as Drosophila and C. elegans . In addition, the echinoderm lineage leading to sea urchins diverged from chordates prior to the large scale gene duplication events that occurred early in the evolution of the vertebrates, and because of this many of the genes that are found as multiple paralogues in vertebrates have only a single homolog in sea urchins. Therefore sea urchins provide a system that avoids the problem of functional redundancies between multiple paralogues that often occurs in vertebrates, and serves as a useful comparison for assessing the significance of conserved genes and regulatory linkages within the genome. Sea urchins cells, like mammalian cells, contain abundant quantities of vaults, which are ~13MDa ribonucleoprotein particles of as yet unknown function [ 2 ]. Vaults are barrel-shaped assemblies composed of multiple copies of three proteins and small untranslated RNA molecules, called vRNA. Intriguingly vaults are up-regulated in certain human multidrug-resistant cancer cell lines, although their role in multidrug resistance remains unclear [ 3 - 6 ]. The high level of conservation among higher eukaryotes of both the major vault protein (MVP) sequence and the barrel-shaped vault structure suggests an important cellular role for the vault. Nucleocytoplasmic transport, sequestration of macromolecules, and protection from xenobiotics have all been proposed as possible functions for the vault [ 4 , 7 - 9 ]. Several recent publications have supported a role for vaults as either a transporter [ 10 , 11 ] or a scaffold protein [ 12 ]. Knock-out mice have been produced lacking one each of the three mammalian vault proteins: MVP; the vault poly(ADP-ribose) polymerase (VPARP); and the telomerase associated protein one (TEP1) [ 13 - 15 ]. These knock-out mice appear to be healthy, indicating that if the vault does perform a critical cellular function there must be a redundant or compensatory pathway in the mouse. Immunofluorescence studies in sea urchins indicate that MVP is present throughout the cytoplasm in cleavage-stage zygotes and in the nucleus in adult somatic cells [ 16 ]. Within the nucleus of coelomocytes, the MVP is present in particularly high concentrations in the nucleolus. This is in contrast to localization studies in most other eukaryotic cells, which show MVP as primarily cytoplasmic [ 17 - 21 ]. During isolation from sea urchins, vaults are found to co-purify with both microtubules and ribosomes [ 22 - 24 ]. The sea urchin MVP cell localization and co-purification results led to a hypothesis that vaults may play a role in nucleocytoplasmic transport of ribosomes and/or mRNA [ 16 ]. The present study provides a comparison of vaults from sea urchins and rats in terms of their molecular composition and MVP protein sequence, as well as by cryoEM imaging and three-dimensional reconstruction. In addition, images obtained by confocal microscopy are presented that indicate differential localization of MVP during sea urchin embryogenesis, suggesting that the highly conserved ribonucleoprotein vault might play a role during development. Results Molecular composition of sea urchin vaults and comparison with mammalian vaults Vaults isolated from mammals, including rats, mice, and humans, are composed of three proteins (MVP, VPARP, and TEP1) and one or more small untranslated RNA molecules, called vRNA. The protein stoichiometry is thought to be 96 copies of MVP with 2–16 copies of VPARP and TEP1 per particle [ 25 ]. The human (99 kDa) and rat (96 kDa) homologs of MVP display 91% similarity. In humans the two high molecular weight vault proteins, VPARP and TEP1, have been sequenced and have masses of 193 and 290 kDa, respectively [ 18 , 26 - 28 ]. vRNA accounts for ~5% of the total mass of the mammalian vault particle. Four human vRNA genes have been identified, encoding vRNAs that range from 86 to 99 bases in length [ 8 , 28 ]. Rat vaults in contrast have only one species of vRNA, 141 bases in length [ 29 ], which appears as a ~37 kDa band by SDS-PAGE [ 25 ]. All of the mammalian vRNA sequences that have been sequenced thus far show ~80% sequence identity and have similar predicted secondary structures [ 18 ]. In rat vaults vRNA can be degraded by a harsh treatment with two RNases leaving the rest of the vault particle structurally intact [ 25 ]. When purified under identical conditions, the molecular composition of the sea urchin vault is more complex than that of the rat vault (Fig. 1A ). For both rat and sea urchin vaults, the strongest protein band is that of MVP at ~100 kDa. We have previously demonstrated that polyclonal antiserum generated against the sea urchin ~100 kDa protein has cross-reactivity with the rat vault MVP, as well as with the Dictyostelium MVPα and MVPβ vault proteins [ 16 ]. SDS-PAGE indicates that the sea urchin vault has two or more high molecular weight components at ~200 kDa, similar to the VPARP and TEP1 bands for rat and mouse vaults [ 9 , 30 , 31 ]. Searches of the S. purpuratus genome identified clear sequence homologs of both VPARP and TEP1 (data not shown). Purified sea urchin vaults also appear to contain vRNA, as demonstrated by the loss of a 26.5 kDa band after RNase treatment (Fig. 1B ). We note that the apparent molecular weight of the sea urchin RNA molecule is smaller than that of the rat vault vRNA, 26.5 vs . 37 kDa. Figure 1 The molecular composition of the sea urchin vault. (A) SDS-PAGE analysis (4–16% acrylamide gradient gel) of isolated sea urchin (S) and rat (R) vaults. The band at ~100 kDa in both lanes represents the major vault protein (MVP). (B) SDS-PAGE analysis of sea urchin vaults both without (-) and with (+) RNase treatment. The 26.5 kDa band in the (-) RNase lane, which is missing in the (+) RNase lane, is thought to correspond to the sea urchin vRNA. Mr represents the molecular weight marker lane. What is noticeably different between the sea urchin and rat vaults is the number of protein bands within the molecular weight range of 14 to 55 kDa in the sea urchin vault that are not observed in the rat vault. Since sea urchin vaults were purified in the same manner as rat vaults, we conclude that either the molecular composition of the sea urchin vault is more complex than that of the mammalian vaults, or that the sea urchin vaults have a more varied composition of molecular cargo. Amino acid sequence of the sea urchin major vault protein To more fully characterize and identify the sea urchin MVP, we used expressed sequence tags (ESTs) and trace sequences from the S. purpuratus genome to assemble the coding sequence of the SpMVP gene (see Methods), which is apparently present as a single homolog per haploid genome. The fact that this sequence was found in several blastula-stage ESTs shows that it is expressed in the embryo. The deduced sequence of sea urchin MVP encodes a 95 kDa protein which is slightly smaller than the 96 kDa rat MVP, with 847 vs . 861 aa. These two MVP homologs exhibit 64% identity and 81% strong similarity (Fig. 2A ). The sea urchin MVP sequence is composed of seven repeats (each 41 to 62 residues) in the N-terminal half of the protein, and a predicted coiled coil region (aa 663–762) in the C-terminal half of the protein (Fig. 2B ). The rat MVP sequence has seven similar repeats of unknown function in the N-terminal half and also has a predicted coiled coil region in the C-terminal half [ 32 , 33 ]. Figure 2 The sequence of the sea urchin major vault protein (SpMVP). (A) Amino acid sequence of SpMVP aligned with that of rat MVP. Identical residues are highlighted in yellow, and similar residues are highlighted in green. (B) Bar diagram of SpMVP. The sequence has seven repeats in the N-terminal half of the protein (R1-R7), each repeat consisting of 41 to 62 aa, spanning residues 32–400; and a predicted coiled coil region in the C-terminal half of the sequence (C.C.), residues 663–762. The positions of two possible nuclear localization sequences (NLS1 470-KKAR, and NLS2 498-KPKR), a putative nuclear export sequence (NES, 792–816), and two probable sumoylation sites that are conserved across six species (S1 K308-VKGE, and S2 K707-AKAE) are indicated. In addition to comparing rat and sea urchin MVP sequences, we used pair-wise alignments to compare the amino acid sequences of several other known MVPs, including the two from Dictyostelium (Table 1 ). These comparisons indicate that the MVP sequence is highly conserved between phylogenetically distant species, and that the similarity between sea urchin and rat MVP sequences is comparable to that among distantly related vertebrates, and also to the intraspecific similarity of Dictyostelium MVPs. Table 1 Differences between MVP amino acid sequences of various species Rat Xenopus Zebrafish Sea urchin Dictyostelium A Dictyostelium B Rat 0 81 (9.38 %) 120 (13.67 %) 112 (12.86 %) 151 (17.34 %) 161 (18.51 %) Xenopus 0 91 (10.50 %) 90 (10.48 %) 124 (14.44 %) 154 (17.91 %) Zebrafish 0 113 (12.96 %) 148 (16.91 %) 165 (18.97 %) Sea Urchin 0 121 (14.07 %) 157 (18.15 %) Dictyostelium A 0 137 (15.91 %) Dictyostelium B 0 Pair-wise sequence alignments of MVPs from each species were used to obtain number of amino acids that are not identical, strongly similar, or weakly similar between each pair of sequences. The numbers in parentheses represent corresponding percent difference. The two Dictyostelium MVPs are designated A and B. The sea urchin MVP sequence was analyzed for potential nuclear localization signals (NLS's) and nuclear export signals (NES's), as well as possible sumoylation sites. Although the PredictNLS server [ 34 ] did not find any putative NLS's, visual examination of the sequence resulted identification of two basic regions with three lysine (K) or arginine (R) residues within a four amino acid stretch that are similar to the smallest consensus sequence of the monopartite type NLS [ 35 ]. Comparison of the sea urchin MVP sequence with the NES logo defined by la Cour et al . [ 36 ], indicates that a region of 25 residues near the C-terminus of protein (aa 792 – 816) has a close resemblance to the NES logo. Within this window we find the three most highly conserved leucine (L) residues (L804, L807 and L809); 3 additional residues matching the most favored residue type in the logo; 6 additional residues matching the second or third most favored residue type; and 3 additional residues matching the fourth, fifth of sixth most favored residue type. The Abgents Sumoylation Calculator predicts five highly probable sumoylation sites in order of probability: K308 (VKGE), K731 (LKAE), K707 (AKAE), K736 (AKIE), and K427 (AKDP) within the sea urchin MVP. With the exception of K427, the amino acid sequence surrounding the lysine acceptor residue follow the consensus sequence, ΨKXE, where Ψ represents a hydrophobic residue, K represents the target lysine, X represents any amino acid and E represents glutamic acid [ 37 ]. Sequence alignment of MVP homologs from six species showed that two of these five motifs (K308 and K707) are conserved. Epitopes are also conserved between the rat MVP and the sea urchin MVP. Previously, we generated and purified a rabbit polyclonal antibody against the MVP that copurified with sea urchin microtubules [ 16 ]. These antibodies were shown to recognize both Dictyostelium and rat MVP [ 16 ]. In this report, we show that these affinity-purified antibodies also recognize the MVP in purified sea urchin vault preparations (Fig. 3 ). Two minor bands of ~80 kDa and 50 kDa cross-react with the anti-MVP antibodies indicating that these may be breakdown products of the 100 kDa MVP. In addition to the antibodies generated against the sea urchin MVP, anti-peptide antibodies were generated against a peptide located at the amino terminus of the rat MVP sequence [ 38 ]. This sequence is approximately 70% identical in the sea urchin and rat MVP sequence and the affinity-purified anti-peptide antibodies bind to purified sea urchin MVP (Fig. 3 ). This observation provides experimental support to relate the assembled coding sequence of the SpMVP gene to the major protein component of the purified sea urchin vault. Figure 3 Western blots of isolated sea urchin vaults. (A) Peptide amino acid sequence of rat MVP (aa 19–35) aligned with Sp MVP (25–41). Anti-peptide antibodies were generated against the rat MVP sequence shown and affinity-purified as previously described [38]. Twelve of the rat MVP amino acids are conserved in the Sp MVP peptide sequence. (B) 150 μg of sea urchin egg extract proteins (lane E) and 10 μg of purified sea urchin vault proteins (lane V) were separated on this Coomassie-blue stained SDS-8% polyacrylamide mini-gel. The arrow shows the position of the 100 kDa MVP in (B, C and D). (C) Alkaline-phosphatase stained western blot showing the migration of the pre-stained protein ladder (lane M: 176.5, 113.7, 80.9, 63.8 (pink), 49.5, and 8.4 kDa polypeptides). Affinity-purified anti-sea urchin MVP antibodies [16] recognize a 100 kDa polypeptide in egg extracts (lane E) and in purified vault preparations (lane V). Asterisks indicate two bands of approximately 80 kDa and 50 kDa that may be breakdown products of the MVP. (D) Affinity-purified anti-peptide antibodies (anti-LDQN, [38]) bind to the 100 kDa MVP polypeptide in purified sea urchin vaults. This peptide antibody appears to bind non-specifically to a large number of polypeptides in the sea urchin egg extracts. Electron microscopy and reconstruction of the sea urchin vault Negative-stain EM and cryoEM images of isolated sea urchin vaults reveal that they have the same overall morphology as mammalian vaults (Fig. 4 ). As was noted in cryomicrographs of the rat vault [ 9 ], sea urchin vaults are occasionally observed to be 'open' at the midsection. One advantage of cryoEM over negative-stain TEM is that a cryomicrograph is essentially a projection image of all of the density, external and internal, within a macromolecular assembly. In cryomicrographs, both sea urchin and rat vaults appear to have molecular contents of varying mass per particle (Fig. 4B , white arrow and [ 9 ]). Figure 4 Negative-stain and cryoEM images of isolated sea urchin vaults. (A) Negative-stain electron micrograph. (B) Cryoelectron micrograph. The black arrow indicates a vault that is opening at the midsection, and the white arrow indicates the dark molecular contents within another particle. The scale bar represents 1,000 Å. Note that the magnification of the cryoEM image (B) is slightly higher than that of the negative-stain EM image (A). In order to characterize the three dimensional structure of the sea urchin vault, a three-dimensional structure was calculated using cryoEM images and single particle reconstruction methods. Only particle images of well-formed, fully closed sea urchin vaults were selected for image processing. Refinement of the cryoEM data set produced a three-dimensional reconstruction at 33 Å resolution (Fig. 5 ). The exterior shape of the sea urchin vault reconstruction is nearly identical to the shape of the rat vault reconstruction with a central barrel section and two protruding caps [ 9 ]. When the sea urchin vault reconstruction is cropped in half the large internal cavity is revealed (Fig. 5B and 5C ). As has been noted for the rat vault, the volume of the internal cavity is large enough to enclose an intact ribosome [ 9 ]. Figure 5 The sea urchin vault reconstruction at 33 Å resolution. (A) The full reconstruction, which reveals that the sea urchin vault has essentially the same exterior structure as rat and mouse vaults. (B and C) The reconstruction is shown cropped along two perpendicular axes to reveal the hollow interior. The crop planes are displayed with the strongest density in red and the weakest density in green. Note that the strongest density is in the "shoulder" region at the top and bottom of the central barrel section. The flat portion of one cap is indicated by an arrow in (B). The scale bar represents 100 Å. CryoEM reconstructions of recombinant and tissue derived vaults often show small holes at the cap/barrel junction [ 9 , 39 ]. The sea urchin vault reconstruction does not show these holes, but they are probably obscured by the low resolution of the sea urchin vault structure. Another subtle difference between the sea urchin and rat vault reconstructions is that less density is observed in the flat portion of the sea urchin vault cap (Fig. 5B , arrow). This is the same region of the vault that was identified as the vRNA binding site within the rat vault [ 25 ]. It is also a region that tends to be variable between vault reconstructions [ 39 ]. Cellular localization of MVP during sea urchin embryonic development Earlier immunofluorescence studies indicated that maternal and zygotic MVP is located in the cytoplasm, whereas adult MVP is predominantly nuclear [ 16 ]. The subcellular location of MVP during embryogenesis was not determined by immunofluorescence. However, cell fractionation and immunoblot analyses of embryos showed that while total amount of MVP remained constant during embryogenesis, MVP became progressively concentrated in the nuclear fractions as development proceeded from the mesenchyme blastula stage to the larval stage [ 16 ]. Here we confirm and extend this result using antibody staining and confocal microscopy to localize MVP subcellularly during embryonic development. The confocal images in Figure 6 demonstrate that sea urchin MVP moves from a largely cytoplasmic distribution in the cleavage stage embryo, to a predominantly nuclear and/or perinuclear location at blastula and gastrula stages. Figure 6 Confocal images of sea urchin embryos at various developmental stages. The cellular distribution of MVP is revealed by staining with an affinity-purified polyclonal antiserum against the sea urchin MVP and secondary staining with Oregon Green-conjugated anti-rabbit antibody (A, D, G, J; left column). The nuclei are stained with DAPI (B, E, H, K; middle column). By merging the MVP and DNA images coincident staining is observed as cyan (C, F, I, L; right column). Note that as development proceeds the cellular localization of MVP progressively shifts from cytoplasmic to nuclear. Discussion In this study we used the sea urchin genome to deduce the amino acid sequence of the sea urchin MVP and characterized the structure of the sea urchin vault particle using cryoEM single particle reconstruction methods. It is known that multiple copies of MVP form the recognizable vault structure, as expression of rat MVP in insect cells leads to the assembly of vaults [ 40 ]. Given the relatively high (81%) sequence similarity observed between sea urchin and rat MVP, it is not surprising that the exterior sea urchin vault structure is quite similar to that of rat vaults [ 9 , 39 ]. In addition to MVP, the rat vault is composed of the high molecular weight proteins VPARP and TEP1, as well as the small vRNA. The electrophoretic analysis of purified sea urchin vault presented here shows protein bands and one RNA band that might correspond to homologs of the mammalian vault components VPARP, TEP1, and vRNA. Moreover, sequence homologs of VPARP and TEP1 are found in the sea urchin genome. SDS-PAGE analysis has indicated that the molecular composition of sea urchin vaults is more complex than that of rat vaults. Protein bands in the range of 14 to 55 kDa are observed that might either be additional vault components or macromolecular contents that are present in high copy numbers within the sea urchin vault. One protein in this mass range (~50 kDa) cross-reacts with MVP antisera and is likely to be a breakdown product of MVP. The identities of the additional proteins will be the subject of future investigations. It is tempting to speculate that the difference in protein composition between sea urchin and rat vaults might indicate that sea urchin vaults are transporting or sequestering a larger assortment of macromolecules. Vaults were originally named because of their similarity to arched cathedral ceilings [ 41 ]. This name may be apt in another sense in that the vault might serve to provide a safe enclosure for its molecular contents [ 2 ]. It has been postulated that mammalian vaults might open under certain physiological conditions to allow encapsulation or release of their molecular cargo. Sea urchin vaults are occasionally observed to be partially open at the midsection in cryomicrographs. Thus sea urchin vaults, as well as mammalian vaults, might be able to regulate what is contained within the large internal cavity. Both sea urchin and rat vaults [ 9 ] appear to have molecular contents in cryomicrographs. However as the features of the contents vary from vault to vault they are inappropriately averaged or "smeared" during the image reconstruction process. A difference map analysis between cryoEM reconstructions of intact and RNase-treated rat vaults led to the localization of the vRNA to the flat portions of the rat vault caps [ 25 ]. The reconstruction of the sea urchin vault reveals a difference in this region of the vault and appears more similar to the RNase-treated rat vault [ 25 ]. Although RNase-treatment does not harm the structure of the rat vault, it does appear to affect the sea urchin vault. Several attempts were made to collect negative-stain and cryoEM images of the RNase-treated sea urchin vaults, but no intact vault particles were observed. The difference observed in the cap density and the differing response to RNase-treatment could imply that the vRNA has a different location or a more important structural role in the sea urchin vault than in the rat vault. The confocal images reveal a progressive enrichment of MVP in the nucleus and perinuclear cytoplasm during sea urchin embryogenesis. Our previous biochemical fractionation studies confirm that the MVP accumulates in the nucleus beginning at the mesenchyme blastula stage [ 16 ]. This important stage of development marks the transition between proliferation and differentiation. As a consequence of differentiation the cells are preparing to exit from the rapid cell cycles that followed fertilization. For example, mRNAs which were abundant in the early cleavage stages, such as cyclin mRNA, decrease dramatically at the blastula stage [ 42 ]. New post-cleavage stage mRNAs begin to accumulate at blastula stage. We suggest that vaults participate in nucleocytoplasmic transport that accompanies remodeling of nuclear architecture in preparation for terminal cell differentiation [ 43 ]. While MVP localization studies in mammalian cells reveal a largely cytoplasmic location, the published staining pattern is consistent with there also being a small amount of nuclear MVP [ 17 , 18 ]. While no classical NLS sequence is found in either the sea urchin or rat MVP, there are basic sequences that might potentially fulfill the requirements of an NLS. The C-terminal region of sea urchin MVP has a region that strongly resembles the NES logo assembled from known NES's [ 36 ] and this region is fairly well conserved in MVP from six species. In addition to possible NLS and NES signals, there are several sumoylation signals within the sea urchin MVP. Sumoylation is a dynamic and reversible process that involves the covalent attachment of the small ubiquitin-like modifier (SUMO) protein to protein substrates. Similar to ubiquination, sumoylation requires four enzymatic steps to attach the carboxyl-terminal glycine of SUMO to the ε-amino group of lysine (reviewed in [ 44 ]). However, unlike traditional ubiquination, sumoylation does not mark the protein for degradation. The wide variety of SUMO targets identified to date does not reveal a single function for this protein modification (reviewed in [ 45 ]). A variety of transcription factors and cofactors, viral proteins, proteins associated with nuclear architecture and genome surveillance, as well as signal transduction molecules appear to by sumoylated [ 44 ]. Importantly for this study, two proteins of the nuclear pore complex, RanGAP1 and Ran-binding protein 2 are both SUMO substrates. Sumoylation of RanGAP1 is important for the nucleocytoplasmic transport of this cytoplasmic nuclear import factor [ 46 - 48 ]. In addition, RanBP2, a docking protein at the cytoplasmic surface of the nuclear protein complex is a SUMO E3 ligase, also known as Nup358 [ 49 - 51 ]. These observations suggest that SUMO plays an important role in nucleocytoplasmic trafficking. In this regard, sumoylation of the major vault protein may regulate the transport of the vault particle through the nuclear pore complex. Taken together these results suggest that the vault may play a role in delivering macromolecules to the nucleus during embryonic development in sea urchins. A trafficking role has also been proposed for vaults in human cells based on the observations that MVP interacts with the estrogen receptor in MCF-7 breast cancer cells [ 10 ] and with the tumor suppressor PTEN in HeLa cells [ 11 ]. Estrogen receptor is involved in regulating eukaryotic gene expression and can affect cellular proliferation and differentiation. Estrogen treatment is observed to increase the association of the estrogen receptor with MVP in the nuclei of MCF-7 cells, thus leading to the hypothesis that vaults may be involved in nucleocytoplasmic shuttling of the estrogen receptor and in modulating the effect of steroid hormones. PTEN negatively regulates the phosphoinositide 3-kinase pathway and cell growth. MVP has been shown by a yeast two-hybrid screen to be a dominant PTEN-binding protein. It has been postulated that perhaps the vault serves to mediate the cellular localization PTEN, thus potentially affecting cell growth. The results presented here suggest that sea urchin vaults contain various macromolecules in addition to the two main vault-associated proteins, VPARP and TEP1. Human MVP is known to interact specifically with the tyrosine phosphatase SHP-2, an enzyme that plays an important role in intracellular signaling [ 12 ]. It has been demonstrated using MVP-deficient mouse fibroblasts that MVP helps to support cell survival. MVP is proposed to act as a scaffold protein for SHP-2 and other extracellular-regulated kinases (Erks) and thus facilitate, or somehow modulate, growth factor signaling. Perhaps the varied macromolecular contents of the sea urchin vault include proteins involved in sea urchin growth factor signaling. Conclusions It would seem likely that the sea urchin MVP is transported to the nucleus for a reason. Presumably cells must expend a considerable amount of energy to achieve this differential localization, as MVP is a relatively abundant protein. The MVP concentration in the unfertilized sea urchin egg is on the order of 10 μM, as determined by immunoblotting [ 16 ]. This maternal store of MVP is sufficient to assemble 10 7 intact vault particles. By comparison, the mature sea urchin egg contains approximately 4 × 10 8 ribosomes [ 52 ]. Although the function of the vault is not yet clearly delineated, our results on the highly conserved structure, molecular composition, and differential cellular localization of MVP suggest that vaults may be important for nucleocytoplasmic trafficking during embryonic development. Methods Isolation of sea urchin and rat vaults Vaults were purified to homogeneity from unfertilized eggs of the Pacific coast sea urchin Strongylocentrotus purpuratus . Animals were spawned and the eggs were collected and washed as described [ 53 ]. Washed eggs (150 ml) were homogenized with a motor driven Potter-Elvehjem-type tissue grinder in an equal volume of buffer (100 mM PIPES-KOH, pH 7.3, 1 mM MgSO 4 , 4 mM EGTA, 2 mM DTT, 1 mM GTP, 0.2 mM PMSF, 10 μg/ml leupeptin and 1 μg/ml pepstatin A). The homogenate was centrifuged at 18 K rpm (~39,000 g) in a Beckman J20 rotor at 4°C for 45 min. The resulting supernatant fluids were decanted into a 500-ml Erlenmeyer flask at room temperature. A fresh aliquot of GTP was added such that the GTP concentration was increased by 1 mM. The DMSO was added in three aliquots with gentle mixing until a final concentration of 15% (v/v) was reached. Microtubule assembly was promoted by incubating the supernatant fluids in a 24°C water bath for 30–45 min. The microtubules were pelleted by centrifugation at 18K rpm (~39,000 g) in a Beckman J20 rotor at 24°C for 30 min. The supernatant fluids (H1S) were carefully removed and drop-frozen in liquid nitrogen and stored in a -80°C freezer. Microtubules in the pellet were purified by two subsequent cycles of assembly and disassembly as previously described [ 54 ] and used for purposes other than the current study. Sea urchin vaults were purified from the microtubule-depleted supernatant fluids that had been previously frozen in liquid nitrogen. Approximately 150 ml of frozen supernatant (H1S) was thawed and mixed with an equal volume of 50 mM Tris-HCL, pH 7.4, 1.5 mM MgCl 2 , 75 mM NaCl, 1 mM DTT and 1 mM PMSF. This pooled supernatant was used as the starting point for the vault purification. This extract was treated as if it was a rat liver homogenate and all subsequent purification steps were as described for rat liver vaults, including purification on sucrose density gradients and two cesium chloride gradients [ 9 ]. Rat vaults were purified from liver by sucrose-density and cesium chloride gradient centrifugation as described by the Rome laboratory [ 9 ]. Molecular composition of sea urchin vaults Vault samples were analyzed by SDS-PAGE with the discontinuous buffer formulation of Laemmli et al ., [ 55 ] and stained with Coomassie Blue R-250 (Fig. 1A ) or silver (Fig. 1B ). Assembly and analysis of the sea urchin major vault protein sequence Trace sequences from the S. purpuratus genome project [ 56 ]were used to assemble the coding sequence of the SpMVP gene. Toward this end the TBLASTN algorithm [ 57 , 58 ] was performed using the rat MVP as a query sequence to search the NCBI trace archive of the sea urchin genome that is now at near 10x coverage. Due to the high level of sequence conservation it was possible to assemble the urchin sequence from the TBLASTN results using the rat MVP as a scaffold. The urchin MVP sequence was assembled in such a way that maximum similarity to the rat scaffold sequence was maintained. The fully assembled SpMVP sequence was then verified by using it to query the NCBI sea urchin EST collection (representing mRNAs expressed in the embryo and larva) by TBLASTN. Except for a small region (A728 to G785) near the C-terminal sequence, the entire SpMVP was covered by S. purpuratus ESTs, with the following NCBI accession numbers: BG784484, BG784394, CD305511, CD295832, CD295616, CD304000, CD310423, CD307400, CD305382, CD292179, CD305646, CD333690, CD309547, CD292477, and BG783194. The gap from A728 to G785 was covered by a sequence in the NCBI trace archives (gnl\|ti\|287010960). The assembled SpMVP sequence has been deposited in the NCBI database under accession number BK005641. We note that the sea urchin MVP has a molecular weight of 95 kDa, calculated from the protein sequence. Previously the sea urchin MVP was referred to as a 107-kDa polypeptide based on its apparent molecular weight by SDS-PAGE [ 16 ]. The program COILS [ 59 ] was used to identify a likely coiled coil region in the C-terminal half of the protein (aa 663–762) [ 60 ]. The sea urchin MVP sequence was submitted to the PredictNLS server [ 34 , 61 ]. No potential NLS sequences were found by this server. The two possible NLS sequences shown in Figure 2B are based on sequence observation. The sea urchin MVP sequence was compared by hand to the NES logo described by la Cour et al . and based on the alignment of 58 high-quality NES's [ 36 ]. The sea urchin MVP sequence was submitted to the Abgent Sumoylation Calculator [ 62 ]. Five motifs with high probability of being sumoylation sites were identified (scores 0.6889 to 0.9278). Two of these five motifs are conserved among MVP sequences of six species ( Strongy. purp., Homo sapiens, Mus musculus, Rattus norv., Danio rerio , and Xenopus laevis ) and are indicated in Figure 2B . The CLUSTALW alignment between sea urchin and rat MVP in Figure 2A was produced by submitting the sequences to the CLUSTALW server [ 63 ]. Negative-stain electron microscopy Isolated sea urchin vaults were applied to freshly glow-discharged formvar-coated copper grids (Electron Microscopy Sciences). Excess liquid was wicked away and the samples were briefly air dried. Samples were stained with uranyl acetate and observed using a JEOL 1200EXII transmission electron microscope. CryoEM imaging and three-dimensional reconstruction CryoEM grids were prepared using cryogenic plunge freezing methods described previously [ 9 , 64 ]. Digital cryoelectron micrographs were collected on an FEI/Philips CM120 transmission electron microscope with a LaB 6 filament, a Gatan 626 cryotransfer holder, and a Gatan slow scan CCD camera (1024 × 1024 pixels, YAG scintillator). One hundred and forty-one cryomicrographs were collected with a nominal magnification of 45,000×, and with two different defocus values (-1.0 and -0.6 μm). The pixel size in the cryomicrographs is 4.1 Å on the molecular scale as determined by calibration with a catalase crystal. The QVIEW software package was used to extract 481 individual vault particle images with a selection box size of 200 × 200 pixels [ 65 ]. The initial translation step was performed by cross-correlating each particle image with an 180° rotated version of itself. The IMAGIC software package was used for all 3D image processing steps [ 66 ]. The published RNase-treated rat vault reconstruction was used as a search model in the first round of refinement [ 9 ]. C8 symmetry was assumed for the sea urchin vault for the first several rounds of refinement, as was used for the intact rat vault reconstruction [ 9 ]. After a few rounds of refinement the two ends of the vault appeared nearly identical, and from then on D8 symmetry was imposed, as was done for the RNase-treated rat vault reconstruction [ 25 ]. The particles images were corrected for the contrast transfer function (CTF) of the microscope using the CTF equation published by Baker et al . [ 67 ]. The following CTF parameters were used: Cs = 2 mm, fraction of amplitude contrast = 0.1, and kV = 120. A subset of 409 particle images that agreed best with the rest of the set were selected for the final reconstruction. The resolution was calculated using the Fourier shell correlation method with an elliptical mask applied to remove noise and disordered contents. The resolution was found to be 33 Å by the 0.5 correlation threshold criterion. The sea urchin vault reconstruction is shown filtered to 33 Å resolution and contoured so that the surface appears continuous. All of the image processing was performed on HP/Digital alpha unix workstations. The graphics representations were produced with the AVS (Advanced Visual Systems, Inc.) software package. Antibody staining and confocal microscopy of whole mounted embryos Staged embryos were fixed in -20°C Methanol for 20 minutes on ice, then washed three times in PBS plus 0.2% Tween-20 (PBST). Embryos were blocked for 30 minutes on ice in PBST plus 5% Bovine Serum Albumin, then incubated with an appropriate dilution of an affinity purified antibody against MVP [ 16 ] overnight at 4°C. Following three washes for 5 minutes each in PBST, the embryos were incubated for 1 hour at room temperature in 1 μg/ml Oregon Green 488 goat anti-rabbit antibody. Embryos were washed three times in PBST and stained in PBST with 300 nM DAPI for 10 minutes at room temperature during the second wash. Digital confocal images were collected using a Leica TCS confocal microscope. Authors' contributions PLS directed the cryoEM and three-dimensional image processing as well as helped to draft the manuscript. MM performed the cryomicroscopy and image processing. JL provided technical assistance. CDS performed the antibody staining and confocal imaging of whole-mounted embryos. AJR assembled the SpMVP sequence and performed the sequence alignments used to calculate sequence similarities and differences. JAC directed the research pertaining to vault localization in the embryo, and drafted Figure 6 and the relevant parts of the manuscript. KAS conceived of the study, participated in its design and coordination, purified the sea urchin vaults, and helped to draft the manuscript. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC550661.xml
515301	Validating internal controls for quantitative plant gene expression studies	Background Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Results Using real-time RT-PCR to study the expression of 10 poplar (genus Populus ) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Conclusion Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.	Background For many years the vast majority of gene expression studies have employed non-quantitative or semi-quantitative RNA gel blots and RT-PCR analysis. Real-time PCR technology has removed many of the difficulties associated with quantitative gene expression studies [ 1 ], and real-time quantitative RT-PCR (qRT-PCR) is rapidly being adopted as a standard method for in-depth expression studies, including studies of alternative splicing, verification of microarray expression results, and molecular diagnostics [ 2 - 5 ]. Real-time qRT-PCR offers a robust means for precisely quantifying changes in gene expression over a wide dynamic range. It is also applicable to experiments where RNA amounts are limiting, such as for micro-dissected tissues. However, selection of an appropriate normalization method is crucial for reliable quantitative gene expression results [ 1 , 6 ]. The purpose of normalization is to correct for non-specific variation, such as differences in RNA quantity and quality, which can affect efficiencies of the RT and PCR reactions. Normalization to total RNA content poses a number of problems. It is difficult to quantify small amounts of RNA, and variation in RT and PCR reaction efficiencies are not accounted for by this method. Similarly, normalization to an external RNA standard is problematic due to RNA instability. The most commonly used method is relative quantitation, whereby gene expression level is normalized to that of an internal reference gene. While this avoids the problems and limitations of absolute quantitation, selection of a proper internal control–gene expressed at a nearly constant level in all tissue samples being investigated–is required. Failure to use an appropriate control gene may result in biased gene expression profiles, as well as low precision. The consequences may be that only gross changes in expression level are declared statistically significant, or that patterns of expression are erroneously characterized. Until recently, internal controls (often referred to as housekeeping or maintenance genes), were selected based on stability of expression in qualitative studies (e.g., visual examination of RNA gel-blots), via low-sensitivity assays such as densitometry of hybridized blots, or via semi-quantitative RT-PCR. None of these will be adequate for identifying reliable internal controls for real-time qRT-PCR. For example, expression profiling via real-time qRT-PCR of 10 commonly used human internal control genes revealed different degrees and patterns of expression among 13 tissue types, and no single gene was a suitable universal control for all tissue types [ 7 ]. Although 18S rRNA is frequently used as an internal control, it is far from ideal. It requires the use of total RNA and random primers for the RT reaction, and is expressed at very high levels; some means for attenuating 18S expression might be needed when weakly expressed genes are studied. In addition, there can be imbalances in rRNA and mRNA fractions between different samples, and 18S is not always expressed at a constant level in all conditions [ 1 ]. Finally, 18S expression levels appear to be affected to a lesser extent by partial RNA degradation than are mRNA expression levels [ 8 ]. Studies in mammalian and microbial systems, where real-time qRT-PCR has been most extensively applied to date, have begun to include evaluations of various housekeeping genes for normalization [ 7 - 11 ]. Vandesompele et al. [ 7 ] recognized the importance of using statistical approaches to selecting the best internal controls for a given set of samples, and developed a procedure to select internal controls based on the mean pairwise variation of a gene from all other tested control genes. The adoption of real-time qRT-PCR methodology is somewhat reminiscent of the introduction of cDNA expression microarrays in that initial microarray studies did not identify differentially expressed genes by a statistical method, but by an arbitrary cut-off value of fold-change [ 12 ]. Similarly, the first real-time qRT-PCR studies have generally normalized expression levels to an internal control that is assumed to be valid rather than one that has been shown to be valid by statistical analysis of data. More rigorous methods will be needed as qRT-PCR is increasingly applied to study of regulatory genes, and for verifying patterns observed in microarray experiments. In this study, we used real-time qRT-PCR to examine the expression of 10 housekeeping genes in a diversity of poplar ( Populus trichocarpa × P. deltoides , cottonwood hybrid) tissues collected at different developmental stages, and at different times of the year. The goal of our studies was to detect changes associated with seasonal development and tree aging for several regulatory genes. We therefore undertook a study to compare the stability of several potential control genes. We found that the genes tested exhibited very different degrees of variation in expression among tissue samples, and that a statistical and graphical method helped us to select the genes best suited for the developmental studies we were conducting. This approach, which is very similar to a classical method used by plant breeders to assess the relative stability in yield of different varieties [ 13 ], can be applied to any gene or set of tissues to identify the most stable internal controls. Results and Discussion Expression profiling of poplar housekeeping genes Ten housekeeping genes that represent different functional classes and gene families were chosen for study. These include ubiquitins, actins, tubulins, cytosolic cyclophilin (peptidyl-prolyl isomerase), translational initiation factor, elongation factor, and rRNA. Searches of the literature revealed that members of all classes have been used as internal controls for studies of plant gene expression using RNA gel blots or RT-PCR assays. Poplar genes belonging to these gene families were identified via searches of the EST database (Table 2 ). The expression level of these genes was determined in eight tissue samples (Table 1 ) collected over a seven month period from mature female poplar trees growing in plantations in Oregon, USA. Within a single experiment, aliquots of the same cDNA synthesis reaction were used for real-time PCR amplification of each of the 10 genes and all gene primer and cDNA combinations were amplified in triplicate in a single PCR run. The entire experiment was then repeated a second time and results combined for statistical analysis. Quantitation via real-time PCR is based on cycle threshold (C T ). C T is the cycle at which a significant increase in amount of PCR product (measured by increase in fluorescence) occurs, generally the middle of the exponential phase of amplification. Mean C T values (average of both experiments) for each gene are given in Table 3 . We had previously used 18S as an internal control for expression studies using these and other tissue samples and had noticed that 18S C T values sometimes varied considerably (data not shown). This may have been largely due to the high abundance of 18S transcripts. Use of 18S as an internal control for studies of genes expressed at relatively low levels required additional dilution of the cDNA templates for 18S amplification relative to the gene being studied. In the present study, the amount of cDNA was the same for all PCR reactions, but 18S primer concentrations were 50 nM, while all other gene primer concentrations were 600 nM. As expected, 18S was the most abundant (lowest C T ) housekeeping transcript; TUA was the least abundant. Statistical analysis of stability of gene expression level We used single-factor ANOVA and linear regression analyses of C T values to examine variation among tissues and RT-PCR experiments. Examination of the distribution of the residual values from ANOVA indicated that assumptions concerning homogeneity of variance and normality of data were adequately met (data not shown). The ANOVA F-test of differences among tissues indicated that five of the genes showed significant variation in expression among the tissue samples. The degree of residual variation, as reflected in the mean square error (MSE), residuals, or coefficient of variation (CV), varied widely. Four genes had CVs below 5%, and two had CVs at or above 25% (Table 3 ). The mean absolute value of the residuals (Fig. 1 ) varied 4.2-fold, from a level of 0.72 for ACT11 to 0.17 for UBQ. To test whether this variation could be due to chance alone, we tested the variation in size of residuals via Levene's test (Levene 1960). The variation among genes was highly significant (P < 0.004), and the difference in residuals between ACT11 and UBQ was also significant based on Tukey's Studentized Range Test and the Bonferroni t-test at the 5% confidence level. The mean expression level for each gene in each tissue sample was regressed against the overall means for the different tissue samples. This overall mean provides an index of RNA quality and quantity for that tissue sample, much as means over test sites provide an index of site fertility in yield trials [ 13 ]. The slope provides an estimate of the degree to which the gene is sensitive to general expression-promoting conditions, and the residuals (deviation from regression prediction) and mean squared residuals (MS-Reg) estimate the degree to which expression of a gene varies unpredictably after linear effects are removed. The residual variation after regression was substantial (Figure 2 ); MS-Reg varied approximately 14-fold (Table 3 ). Assuming that both constancy over tissues (low slope) and high predictability (low CV) are desired, we created a stability index as the product of slope and CV. The genes with the lowest stability index will usually provide the best controls. In this study, UBQ had the lowest stability index, a result of both a very low slope and very low CV. Selection of internal controls In addition to constancy of expression level, the expression level of an internal control compared to that of the genes being analyzed might be important to consider in certain cases. In our study, two of the most stably expressed genes represented opposite ends of the spectrum. UBQ is highly expressed (mean C T = 15.8), whereas TUA is expressed at a much lower level (mean C T = 28.9) (Table 3 ). For the samples we tested, the high stability of UBQ and TUA expression indicate that use of either as a single internal control gene is appropriate. However, for some studies, no single gene may be adequate. In these cases, a method for normalization to two or more of the most stable internal control genes identified might be necessary. For example, normalizing to the geometric mean of selected internal control genes [ 7 ]. A potential strategy to avoid the additional expense and labor of using multiple internal control genes is to design a PCR primer pair that will amplify two or more members of a control gene family, whose combined expression level may exhibit the desired expression level and stability. Our primers were designed based on a limited EST set that likely did not include all family members, and ESTs vary in sequence quality. Thus, primers could have amplified more than one family member or primer mismatches due to EST sequence errors could have lowered PCR efficiency. Although gel and real-time PCR dissociation curve analyses did not indicate that multiple genes were amplified with our primer sets, these analyses might not detect multiple amplicons from different family members that are the same size and have the same PCR efficiency. As discussed above this is not necessarily a detriment–amplification of multiple family members might result in a more stable internal control than single gene amplification. In addition, the upcoming release of a large poplar unigene set and annotated genome sequence [ 14 ] will improve gene selection and primer design capabilities. Conclusions Using ANOVA and linear regression analysis, we demonstrated that levels of expression stability among a number of potential control genes can vary widely, and that it is not difficult, costly or labor-intensive to test a number of genes. Moreover, such validation tests might have the additional benefit of revealing technical problems, such as excessive variability in RT and PCR efficiency due to RNA quality or inconsistent pipetting. For some experiments, choice of an internal control is straightforward. For example, a number of housekeeping genes should be satisfactory controls for comparisons of transgene expression level in the same tissue type from different transgenic lines grown under identical conditions. However, this is not the case for studies that compare gene expression among different tissue or cell types, at different developmental stages, or under different environmental conditions, as were represented in our study of trees in field environments over a period of seven months. For such studies, internal controls should be carefully tested and validated. Statistical confirmation of internal controls for qRT-PCR should enable previously indiscernible small changes in expression level to be reliability detected. Methods Tissue collection and RNA extraction Tissues were collected from five or six year-old ramets (genetically identical trees) of a single female poplar hybrid clone ( P. trichocarpa × P. deltoidies ) over a seven-month period in 2001 (Table 1 ). The trees had been growing in commercial plantations in the Columbia River basin northwest of Portland, Oregon USA. Bud scales were removed and tissues were frozen in liquid N 2 and stored at -80°C until RNA extraction. Total RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, CA, USA) with modifications. Tissues (0.2 g) were ground to a fine powder with mortar and pestle in liquid N 2 . The powder was added to a tube containing 1 ml of RNeasy RLT buffer and 0.01 g soluble polyvinylpyrrolidone (PVP-40; Sigma, St. Louis, MO, USA), and homogenized using a polytron for approximately 30 sec. Four volumes of 5 M Potassium acetate, pH 6.5 was added to the homogenate, the mixture was incubated on ice for 15 min, and the precipitate removed by a 15 min centrifugation (12,000 rpm) at 4°C. Supernatant was transferred to two 1.5 ml microcentrifuge tubes and 0.5 volume of 100% EtOH was added. Samples were transferred to RNeasy mini columns and the remaining steps were as directed by the manufacturer's instructions for plant RNA isolation (steps 6–11). RNA was quantified using spectrophotometric OD 260 measurements and quality was assesed by OD 260 / OD 280 ratios and by electrophoresis on 1% formaldehyde agarose gels followed by ethidium bromide staining. RNAs were stored at -80°C. Selection of poplar sequences and PCR primer design To identify poplar homologs of genes commonly used as controls for plant gene expression studies, we queried poplar EST databases with Arabidopsis protein sequences using TBLASTN [ 15 ]. Selected poplar ESTs were then used to query the Arabidopsis protein database using BLASTX (Table 2 ). Primers were designed using Primer3 software [ 16 ] or Primer Express (Applied Biosystems, Foster City, CA, USA) with melting temperatures of 59–60°C. By comparison to related poplar EST sequences, primers were designed to be as specific as possible for the selected gene family member. All primer pairs were initially tested via standard RT-PCR using the same conditions as described below for real-time RT-PCR. Amplification of single products of expected size was verified by electrophoresis on 3% agarose-1000 (Invitrogen, Carlsbad, CA, USA) and ethidium bromide staining. Real-time RT-PCR Contaminating DNA was removed from RNA samples using the DNA-Free kit (Ambion, Austin, TX, USA) according to the manufacturer's protocol, and two-step real-time RT-PCR performed. cDNA was synthesized from 5 μg of RNA using the SuperScript first-strand synthesis system for RT-PCR (Invitrogen) with random hexamer primers according to the manufacturer's instructions, except that the initial 65°C denaturation step was omitted. The cDNAs were diluted 1:5 with nuclease-free water. Aliquots of the same cDNA sample were used with all primer sets for real-time PCR, and amplification reactions with all primer sets were performed in the same PCR run. Reactions were done in a 25 μl volume containing 600 nM of each primer, 6.5 μl of cDNA sample (≈320 ng of input RNA) and 1X SYBR Green PCR master mix (Applied Biosystems). For 18S amplification, primer concentration was 50 nm. Real-time PCR was performed on the ABI Prism 7700 Sequence Detection System (Applied Biosystems) in a 96-well reaction plate using the parameters recommended by the manufacturer (2 min. at 50°C, 10 min. at 95°C and 40 cycles of 95°C for 15 sec and 60°C for 1 min.). Each PCR reaction was performed in triplicate and no-template controls were included. Specificity of the amplifications was verified at the end of the PCR run using ABI Prism Dissociation Curve Analysis Software. The entire experiment, including both the RT and real-time PCR steps, was repeated, giving a total of two experimental replications. Statistical analyses Results (C T values) from the ABI PRISM 7700 Sequence Detection System were analyzed in Microsoft Excel. Single factor ANOVA and regression analysis using the least squares method were performed using the Excel Analysis ToolPak. Assumptions concerning homogeneity of variance and normality were evaluated from inspection of residuals (the difference between an observed value and overall mean for all genes) from the ANOVA (Fig. 1 ). The level and significance of the difference between gene expression levels in different samples were evaluated by Fisher's F statistic [F = between-tissue-sample mean square / error mean square] assuming the three replicate PCR reactions approximated variance between fully independent observations. Other statistics are as defined in Table 3 . The general procedure for data analysis to compare genes for use as internal controls was: 1) Generate data from multiple analyses of gene expression via quantitative RT PCR that can be assumed to be statistically independent (or nearly so), including from multiple independent samples that bracket the experimental conditions of interest. 2) Conduct ANOVA to examine the extent of variation among samples, and (optionally) test their significance using appropriate F-ratios. Examine plot of residuals vs. mean expression level, or use a statistical test, to check normality of data. 3) Genes showing high variance among tissues in ANOVA, especially if accompanied by large mean square errors (or coefficients of variation), are to be avoided as controls. 4) Calculate mean expression level over all genes studied for each sample type as an index of both experimental and biological conditions that promote high levels of measured expression. Use of a large number of genes and tissue samples (e.g., at least five, and preferably many more) are desirable where estimates of stability are to be compared between studies. 5) Regress mean expression level for each gene in each sample type over the mean for the sample type. The estimated slope and mean square residual (deviation from regression prediction) provide estimates of the degree to which the gene is sensitive to general expression-promoting conditions (slope) and whose expression continues to be difficult to predict (residual). 6) Assuming that both constancy over sample types (low slope) and high predictability (low coefficient of variation) are desirable, a stability index can be created as their product (or via other mathematical means), and the gene with the lowest value chosen. 7) Alternatively, visually inspect regression and residual plots to select genes that would be most suitable as controls for specific sets of experimental conditions. Authors' contributions AMB developed the molecular methods, participated in design of the study, and drafted the manuscript. IAY designed primers, carried out the real time RT-PCR experiments, performed the statistical analysis and drafted the manuscript. SHS conceived the analysis, and participated in its design. All authors read, helped to edit, and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC515301.xml
538278	DEDB: a database of Drosophila melanogaster exons in splicing graph form	Background A wealth of quality genomic and mRNA/EST sequences in recent years has provided the data required for large-scale genome-wide analysis of alternative splicing. We have capitalized on this by constructing a database that contains alternative splicing information organized as splicing graphs, where all transcripts arising from a single gene are collected, organized and classified. The splicing graph then serves as the basis for the classification of the various types of alternative splicing events. Description DEDB is a database of Drosophila melanogaster exons obtained from FlyBase arranged in a splicing graph form that permits the creation of simple rules allowing for the classification of alternative splicing events. Pfam domains were also mapped onto the protein sequences allowing users to access the impact of alternative splicing events on domain organization. Conclusions DEDB's catalogue of splicing graphs facilitates genome-wide classification of alternative splicing events for genome analysis. The splicing graph viewer brings together genome, transcript, protein and domain information to facilitate biologists in understanding the implications of alternative splicing.	Background The completion of the draft sequence of the Drosophila melanogaster genome in March 2000 [ 1 , 2 ] and the availability of quality annotations by FlyBase in 2002 [ 3 ] presents an excellent opportunity for the study of alternative splicing. Although the annotations themselves provide an insight to the amount of alternative splicing, they do not provide any classification of the types of alternative splicing events present. Different forms of alternative splicing have different biological bases and the classification of alternative splicing events is critical for further work in deciphering the regulatory controls that govern these processes. To this end, we transformed all known gene structure information obtained from the genome annotations into splicing graphs based on the approach first proposed by Heber et al. in 2002 [ 4 ]. We then created simple but robust rules for classifying the splicing graphs into various alternative splicing events. The rules created allows for the detection of multiple forms of alternative splicing within the same gene. To facilitate the assessment of the impact of alternative splicing on the protein product in particular with respect to the domain organization of the protein, Pfam [ 5 ] domains were mapped onto the transcripts using HMMER [ 6 ]. All these data were then loaded into DEDB ( Drosophila melanogaster Exon Database) [ 7 ]. To aid in visualizing these splicing graphs, a web-based splicing graph viewer was also developed. The splicing graph viewer integrates gene structure, transcript, protein and domain information into an easily understandable interface that is viewable with any current web browser. The splicing graphs as well as the alternative splicing event classifications are available for download as XML files. A XML schema is available for parsing and validation of the XML files. Construction and content Data source Drosophila melanogaster genome annotations (release 3.2) were obtained from FlyBase [ 8 ] as Game XML files. Gene structure information including the location of the transcript, the start and end positions of each exon that make up the transcript and the protein coding region were parsed out, checked for consistency and then loaded into a relational database (MySQL). Pfam HMM models were retrieved from Pfam release 12 and used as the database for the hmmpfam program (part of HMMER) to search the transcript protein sequences for structural domains, with an expectancy values of less than 0.001. The results of the search were parsed, mapped onto the protein sequence and imported into the database. Construction of the splicing graphs The transcripts contained in the database were retrieved and clustered on the basis that they occupy overlapping genomic positions. Splicing graphs are then constructed using these clusters of transcripts. In each cluster, exons and introns having identical start and end positions are merged into nodes and connections respectively. The nodes are then linked via connections to form the complete splicing graph that is loaded into the database. In cases where the transcripts are located on the negative strand, the entire splicing graph is reverse complemented so that all the splicing graphs contained in the database have sense strand nucleotide sequences. These steps are graphically depicted in Figure 1 . The result of this process generated 13,222 splicing graphs of which 2,646 are alternatively spliced. Details of the contents of the database are found in Table 1 and on the website via the "Stats" link. Classification of alternative splicing Rules are then derived to detect specific alternative splicing events as shown in Figure 2 (details and examples of the rules are available on the website). Apart from the classical alternative splicing events like cassette exons, intron retention, alternative donor sites and alternative acceptor sites, we have also elected to classify other gene structure events like alternative transcriptional start/termination sites as well as alternative initiation/termination exons. The reason for the existence of the alternative initiation/termination exon categories is due to the fact that the 5' and 3' ends of the transcripts are usually difficult to determine experimentally and are thus less accurate. Therefore, any differences in the start and end of the transcripts could be simply due to the sequencing difficulties. The inclusion of the alternative initiation/termination exons category is an attempt to circumvent this problem as alternative initiation/termination exons (which are classified based on the end position of initiation exons and the start position of termination exons) are unaffected by the sequencing difficulties and thus represent true alternative exons. Alternative transcriptional start/termination sites, however, are dependent on sequencing results and provide a means of classifying gene segments with differences in the start positions of initiation exons and the end positions of termination exons, with a view to updating entries in this category, when the 5' and 3' ends of these transcripts are determined accurately. These rules were then used on all the splicing graphs and the detected alternative splicing events loaded into the database. Access The database together with the splicing graph viewer is freely available at . Users can query the database using FlyBase gene names, FlyBase Gene Symbols, Pfam Accession Numbers or Pfam Identifiers via the query page. Users can also query the database using BLAST [ 9 ] searches. This is particularly useful if one wishes to know the Drosophila melanogaster homology together with alternative splicing information of a particular gene. Lists of splicing graphs for the various types of alternative splicing events are also provided on the website for users who are interested in a certain type of alternative splicing. For users who wish to use large subsets of the data, they can download the XML files available from the same site. To aid parsing and validation of the XML file, a XML schema is available. DEDB can also be accessed via links on Flybase gene records, under the external database links section. Correspondingly, the DEDB Splicing Graph Viewer provides links back to FlyBase Gene and Annotation records, where experimental evidence for the gene structure has also been provided. Basic statistical analysis of the database can be found at the DEDB website . Splicing graph viewer The splicing graph viewer consists of HTML pages created using a series of Python CGI (common gateway interface) scripts served by the Apache web server. The splicing graph viewer (Figure 3 ) is a three frame HTML page that shows the splicing graph in the center frame with detailed textual information in the bottom frame and navigation aids in the top frame (details elaborated on the website). The content is organized such that generalized information is provided to the users initially, permitting users to quickly zoom in on the information they need by clicking on an item of interest. The splicing graph shown in Figure 3 has alternative acceptor sites, where nodes 1 and 3 are alternatively used. Each node here can be selected and the corresponding node information, with sequence details is then dynamically displayed in the bottom frame. The transcripts leading to this splicing graph are depicted below the splicing graph. The connections in each transcript represent introns, which can also be selected to obtain intron-specific information. The rationale for providing the transcripts is because not all the paths in the splicing graphs are expressed transcripts, so the connections depicted in the splicing graph view are transcript-specific and thus not selectable by DEDB users. The provision of schematic diagrams of the transcripts along with the splicing graph provides the user with knowledge of which transcripts are expressed/detected. Below each transcript, links to structural domains, are shown as thin lines wherever available, linked to detailed information derived from Pfam and viewable on the bottom frame. The sequences displayed by clicking on the nodes in the splicing graph are always shown in the sense orientation to facilitate translation of coding sequences and Pfam mapping, while the exon and intron sequences shown by selecting genomic segments on the transcripts will retain their original orientation identified by chromosomal mapping. Utility and Discussion Visualization of alternative splicing By condensing all the various splicing variants into a single graph, where each splicing variant is a path through the graph, users can quickly establish the types and effects of various alternative splicing events present in the gene. Users can quickly pick up bifurcations which denote alternative splicing events far quicker than in the case of the traditional approach of presenting separate schematic representations of each splice variant, where the user has to correlate the splicing patterns from the transcript diagrams, to determine the impact and type of alternative splicing. The classical approach is particularly tedious in cases where the number of splice variants are numerous (for example the Drosophila moe gene, splicing graph 916, on the DEDB methodology link) resulting in the user having to correlate large amounts of data to comprehend all the alternative splicing events taking place. The DEDB schematic representation of the splicing graph is different from the one proposed by Heber et al. [ 4 ] and implemented in Alternative Splicing Gallery (ASG) [ 10 ]. The original representation used single linear block representation of exons connected by lines representing introns and alternative donor and acceptor sites as well as intron retention represented by single blocks. Instead, we have chosen to depict all the exons individually as we felt that this is more intuitive for biologists by making the impact of the alternative splicing more pronounced. Protein sequence details like the start and end of translation as well as detected Pfam domains are presented by the splicing graph viewer. This allows biologists to infer the impact of alternative splicing on the corresponding protein sequences as well as the domain organization. Users can also download FASTA sequences of specific entities like introns and exons for other analysis. Biologists may also be interested in the Drosophila melanogaster homology of their gene of interest, which is made possible through a BLAST search on the DEDB query page. The splicing graph of the Drosophila melanogaster homology may provide insights into the possible splice variants in the gene of interest. It could also provide information on the level of conservation of alternative splicing between orthologs. Classified splicing dataset The use of splicing graphs allows the creation of simple but robust rules that can detect multiple distinct alternative splicing events within the same gene. Traditional approaches usually require the construction of more complex rules. For example, the detection of a cassette exon in the tradition approach requires that an internal exon be checked against all the introns in all the splicing variants to detect instances where the exon falls within an intron. This process has to be repeated for each exon against all the introns resulting in a long and complex computation. Furthermore as the exon could be found in several splicing variants, the detected cassette exon could be redundant and additional steps have to be taken to remove this redundancy. All of this is avoided by the splicing graph representation, as it is a condensed view of all the various splicing variants arising from a single gene. Classification of the alternative splicing types in Drosophila melanogaster would allow users to target specific types of alternative splicing events for analysis. This is useful as the various types of alternative splicing have different biological bases and therefore exhibit different phenotypes. The analysis of these phenotypes will be greatly aided by a set of data that is specific to one form of alternative splicing as provided by DEDB. The availability of a clean datasets of alternative splicing events [ 11 , 12 ] has proved to be useful in providing insights into the phenomenon of alternative splicing [ 13 ]. The data available from DEDB would no doubt be useful to many users studying alternative splicing as a major factor leading to complexity in higher eukaryotes. Initial analysis A summary of the alternative splicing events in DEDB is presented in Table 1 . Detailed statistical information (general statistics, exon and intron length statistics and motif analysis) are available from the "Stats" page of the website (Lee, Tan and Ranganathan, unpublished results). Note however that the genes models are constructed with far more 5' ESTs than 3' ESTs [ 3 ] and the results must be viewed in the light of available experimental EST data. Of the total of 13,222 genes in DEDB, 2,646 (20%) are alternatively splicing. This is significantly less than the amount of alternative splicing found in higher eukaryotes like humans [ 14 ], but sufficient to indicate that alternative splicing is a common phenomenon in Drosophila melanogaster . The amount of alternative splicing increases to 24.4% if we consider transcript diversity in the 10,848 multi-exonic genes alone. Failure of intron definition is more likely to result in intron retention as opposed to exon definition in which case, failure leads to cassette exons. Initial analysis of the DEDB data indicates a bias towards cassette exons (1,228) over intron retention (983) events, so that exon definition is less stringent than intron definition. The short introns in Drosophila melanogaster are also thought to result in greater intron definition. The data observed could be due to the splicing machinery adopting a definition model dependent on the length of the intron or exon in question [ 15 ]. This is supported by the fact that cassette exons tend to be flanked by introns far longer than the mean value (exon and intron length statistics available via the "Stats" link). The median for the cassette exon length is 150 bp in contrast with the flanking 5' and 3' introns, which are 653 bp and 639 bp respectively. The reverse is also true for intron retention where the median for the intron being retained is 101 bp while the flanking 5' and 3' exons are 163 bp and 261 bp respectively. Information content analysis indicates that alternative donor and acceptor sites (with mean values of 5.95 and 5.61 bits) possess less information than constitutive sites (9.74 and 8.52 bits respectively; additional data available on website). This observation is consistent with the general notion that alternatively spliced exons exhibit splicing motifs deviating more from the consensus motifs [ 16 ]. Cassette exons (CE) and intron retentions (IR) also show lower mean individual information content on both donor (CE: 6.76 and IR: 5.39 bits) and acceptor sites (CE:7.08 and IR: 6.19 bits) as compared to constitutive exons. The addition of Pfam domain information allows users to assess the impact of alternative splicing events on the proteins generated, enabling correlations not possible with the genome annotations alone. Future work Future work would focus on integrating other relevant information onto the splicing graphs, such as three-dimensional structural information as well as DEDB analysis results. Expansion of the splicing graph representation available in DEDB to other organisms is also underway. Conclusions The data housed in DEDB is organized as splicing graphs, which allows for ease of alternative splicing classifications. This has allowed DEDB to provide clean sets of data containing specific types of alternative splicing events. These specific sets of data could prove useful in understanding the biological basis of alternative splicing because different forms of alternative splicing have different biological basis. The splicing graph viewer provided allows biologists to quickly and intuitively understand the effects of alternative splicing on a gene of interest, thus aiding their research. Availability and requirements The database is available at suitable for most graphical web browser. XML files of the data contained in the database are also available together with an XML schema to aid parsing. Authors' contributions BTKL carried out the construction of the database as well as the splicing graph viewer. TTW and SR are responsible for the database concept and participated in its design and construction. All authors have read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC538278.xml
538287	An adaptogenic role for omega-3 fatty acids in stress; a randomised placebo controlled double blind intervention study (pilot) [ISRCTN22569553]	Background There is evidence for an adaptive role of the omega -3 fatty acid, docosahexaenoic acid (DHA) during stress. Mechanisms of action may involve regulation of stress mediators, such as the catecholamines and proinflammatory cytokines. Prevention of stress-induced aggression and hostility were demonstrated in a series of clinical trials. This study investigates whether perceived stress is ameliorated by DHA in stressed university staff. Methods Subjects that scored ≥ 17 on the Perceived Stress Scale were randomised into a 6-week pilot intervention study. The diet reactive group was supplemented with 6 g of fish oil containing 1.5 g per day DHA, while the placebo group was supplemented with 6 g a day of olive oil. The groups were compared with each other and a wider cross sectional study population that did not receive either active or placebo intervention. Results There was a significant reduction in perceived stress in both the fish oil and the placebo group from baseline. There was also a significant between-group difference between the fish oil group and the no-treatment controls in the rate of stress reduction ( p < 0.05). However, there was not a significant between-group difference between the fish oil and the placebo group, nor the placebo group and the control group. These results are discussed in the context of several methodological limitations. The significant stress reductions in both the fish oil and the placebo group are considered in view of statistical regression, an effect likely to have been exaggerated by the time course of the study, a large placebo effect and the possibility of an active effect from the placebo. Conclusion There were significant differences ( p < 0.05) in the fish oil group compared with no-treatment controls. This effect was not demonstrated in the placebo group. As a pilot study, it was not sufficiently powered to find the difference between the fish oil group and the placebo group significant. Further work needs to be undertaken to conclusively demonstrate these data trends. However, the findings from this research support the literature in finding a protective or 'adaptogenic' role for omega-3 fatty acids in stress.	Background Rousseau and Moreau et al [ 1 ] demonstrated an ameliorated cardiac response to a mild socio-social stress in DHA (the omega-3 fatty acid, docosahexaenoic acid) fed rats. The feeding schedule induced mild increases in heart rate in the sunflower oil fed group but not the DHA group. A corresponding increase in norepinephrine was significant only in the sunflower oil group. DHA also decreased systolic and diastolic blood pressure. The beneficial cardiovascular alterations, evident within a few weeks of supplementation, corresponded with high cardiac phospholipid membrane levels of DHA found on post-mortem examination. Mills and Prkachin et al [ 2 ] found an effect from borage oil (found to rapidly increase membrane dGLA, the omega-6 dihomogammalinolenic acid) but not fish oil (rich in DHA) in cardiac parameters of stress reactivity in humans. Unfortunately, the potential confounds were not adequately discussed, bringing into question the reliability of these results. For instance, there was no mention of subject withdrawals or dropouts, a flush-out period, background diets, or background stress levels. Chronic stress levels have subsequently been demonstrated to influence reactivity to an acute stressor [ 3 ] and the effectiveness of DHA to reduce stress [ 4 ]. A research group in Japan have shown a protective effect of DHA during stress. A multi-centred randomised, placebo-controlled, double blind study involving 53 medical students and 3 months supplementation with 1.5 g/d was timed to coincide with a period of intense stress. They found that aggression towards others was significantly increased in the control group by 8.9% from baseline ( p < 0.007) during the final examinations. There was no difference in aggression in the DHA group [ 5 ]. DHA prevented an increase in aggression during the examination period. The second study was modelled on the previous study with the major difference being timing [ 6 ]. A similar but non-stressed sample 46 of university students were tested for aggression. The second study was designed to not coincide with any periods of academic stress. It commenced at the start of the summer holidays. The researchers found that DHA does not affect aggression of normal volunteers under non-stressful conditions. Hostility was also found to increase significantly during psychological stress[ 7 ]. In a randomised, placebo-controlled, double blind study, 41 students took either 1.5 g/d DHA or placebo (soy oil) for 3 months. Hostile responses were significantly increased by from 27% (baseline) to 92% (during exams) in the control group, where there were no significant changes in the DHA group ( p < 0.01). There were highly significant between-group differences ( p < 0.002). The same researchers demonstrated that hostility levels significantly decreased in a population of university staff taking DHA supplementation compared with no change in hostility levels in subjects taking the placebo [ 7 ]. DHA appears to have an adaptive effect on hostility. Sawazaki and Hamazaki et al [ 8 ] investigated the effect of DHA on various physiological parameters during psychological stress. Fourteen medical students took either 1.5 g/d DHA or placebo (47% olive oil, 25% rapeseed oil, 25% soy oil and 3% fish oil) for 9 weeks, culminating in a period of intense stress. While there were no significant differences between groups in epinephrine, cortisol, glucose or insulin, DHA significantly reduced plasma norepinephrine (NE) concentrations from baseline (-13%, p < 0.03). This reduction corresponded with a 78% increase in the ratio of epinephrine (E) to NE in the DHA group ( p < 0.02). The higher E:NE ratios were interpreted as a favourable adaptive response to stress. This claim was substantiated by citations of studies which reported a failure to normalise the E:NE ratio during psychological stress observed in patients with duodenal ulcer. This ratio is believed to be protective as it has been associated with lower death rates in 412 older men. Thus, the authors concluded, a possible adaptive mechanism for DHA during stress may be to regulate the E:NE ratio. Another possible mechanism whereby omega-3 fatty acids may be protective in stress is by modulation of proinflammatory cytokines. Maes and Christophe et al [ 9 ] found that exam stress in 27 university students significantly increased the stimulated production of many proinflammatory cytokines ex vivo . Subjects with low serum omega-3 fatty acid levels had significantly higher stimulated production of interleukin-6 at baseline compared with the subjects with high serum levels ( p = 0.026) and a trend towards a significant difference during academic stress ( p = 0.1). Stimulated production of interferon-γ and tumour necrosis factor-α was significantly greater in subjects with low serum omega-3 fatty acids ( p = 0.02). The higher serum omega 3 levels were believed to be protective in academic stress because they were associated with lower levels of pro-inflammatory cytokines The primary aim of the present study is to investigate whether manipulation of dietary fats has an effect on perceived stress, as measured by the Perceived Stress Scale (PSS). The hypothesis is that DHA will ameliorate stress in moderate to highly stressed university staff. Methods A small intervention study was nested within a larger prospective cross sectional study. Figure 1 illustrates the research design. The cross sectional study compared three stress measures, and correlated the respective measures with measures of mood and dietary fats intake [ 10 ]. This was repeated after a 10-week interval. Figure 1 A synoptic overview of the research design showing the smaller intervention study nested within the larger cross sectional study. Note: PSS = Perceived Stress Study [11]; VAS = visual analogue scale; OSI-R = Occupational Stress Inventory-Revised [21]; PANAS=Positive and Negative Affect Scales [22]. Moderately stressed subjects were randomised into a 6-week intervention study. The nutritional intervention study was designed as a double blind randomised placebo-controlled clinical trial (pilot), involving three groups. The three groups were (1) active (1.5 g/d DHA from fish oil); (2) placebo (olive oil); and (3) control (no treatment). The supplementation period was 6-weeks. Study Population All procedures and processes were subject to the prior approval of the Human Research Ethics Committee at Southern Cross University. Sample size A power calculation was conducted using the variability data on the PSS [ 11 ]. The α and β values were set at 0.05 and 0.8, respectively. The resulting sample size requirement was for 50 subjects in each arm to demonstrate a change of 20%, 70 subjects to demonstrate a change of 15% and 175 subjects to demonstrate a change of 10%. For logistical reasons (the research was part of an honours project), the sample size for the study was chosen to be 15 subjects in each arm. Whilst inadequately powered, it was hoped that there were enough subjects to provide data that may indicate data trends. Recruitment All staff members of Southern Cross University were invited via intra-staff email to participate in the study on the effects of dietary fats in stress. Staff that responded to the initial recruitment email had the questionnaire personally delivered. They were instructed to complete the questionnaire and return it via internal mail. Staff members were contacted by phone and invited to participate in the nutritional intervention study if the score on the PSS was greater than or equal to 17. Time course for recruitment and study Recruitment for the intervention study commenced in June 2002 and was conducted over a 4-week period. Scores from the PSS at Time 1 formed the screening for the intervention study. The PSS was re-administered at baseline. The supplementation period commenced at the end of June and was completed by mid July 2002. Time 2 questionnaires were then sent out to all subjects that had participated at Time 1. Questionnaires were returned and scored in September 2002. Inclusion/exclusion criteria Subjects were included if they were 18–60 years, had not taken a course of fish oil in the past three months, refrained from taking other nutritional supplements and/or aspirin and from radically changing their diet for the duration of the trial, and had a normal physical examination. Subjects were excluded for medical history of coronary heart disease, any type of clotting disorder, clinically diagnosed depression, psychiatric history, diabetes mellitus, or in female subjects, pregnancy or lactation. Suitable candidates undertook a brief clinical assessment. Randomisation and blinding Subjects gave informed consent for the study and were subsequently randomised into two groups. A computer program was used to generate the stratified randomisation schedule. The investigators involved with the study had no knowledge of the details or results of randomisation, the study participants had no knowledge of the group to which they had been allocated, all investigators and statisticians associated with the research were blinded regarding ongoing results. The Nutritional Intervention Each capsule contains 1000 mg tuna oil, with 10 mg d-alpha-Tocopherol (vitamin E). The tuna oil was standardised to contain docosahexaenoic acid (DHA) 252 mg per 1000 mg, and eicosapentaenoic acid (EPA) 60 mg per 1000 mg oil. The placebo capsules contained 1000 mg olive oil, consisting predominantly of monounsaturated fatty acids. The placebo capsules were identical to the DHA capsules in every way, including size, shape, colour and smell. There was a 2-week wash out period prior to the commencement of the supplementation period that applied to subjects taking any form of natural or complementary medicine. All subjects were instructed to take 3 capsules with breakfast and 3 for dinner for 6 weeks. Compliance was measured by collecting the bottles with any remaining supplements at the completion of the study. The number of remaining supplements was divided by the total number of supplements dispensed. Less than 85% compliance resulted in the withdrawal of subject data from analysis. Outcome The primary outcome was the differences between groups in changes over time in perceived stress, as measured by the Perceived Stress Scale (PSS) [ 11 ]. The PSS-10 consists of 10 questions designed to measure subjective appraisal of life stress, taking into account appraisal of the ability to cope with the stress. It has adequate reliability and validity as a stress appraisal (perceived stress) measure [ 12 ]. Scores on the PSS have been correlated with other mental and physical health outcomes [ 13 ]. Statistical (multi-level) analysis A two-level structure was used, where level-one units were measurement occasions, consisting of Time 1 and Time 2. Level-two units were the treatment groups, consisting of T1 =the group of subjects that did not go into the intervention study; T2 = the placebo (olive oil) group and T3 = the active (fish oil) group. Results Study population Response rate to the recruitment email was 13.6% of full-time equivalent university staff. The response rate for the return of personally delivered questionnaires was 85%. The flow of participants through the intervention study is given in figure 2 . There were 47 staff members that scored ≥ 17 on the PSS. After the phone screen, 39 staff members were invited for an interview, which involved further inclusion/exclusion criteria, a clinical assessment, and obtaining informed consent. Figure 2 Flow of participants through each stage of the recruitment process and intervention study. A further 11 potential subjects were excluded as a result of the interview process; 9 because they did not want to cease other complementary medicines for the duration of the trial, 1 because of positive findings in the clinical assessment and 1 was a female subject still occasionally lactating. Withdrawals A high percentage (30%) of subjects that began the intervention study were withdrawn; 3 subjects were lost to follow-up and 6 discontinued intervention; 5 due to illness related reasons and 1 because taking the supplements caused discomfort with swallowing. This resulted in 70% (n = 21) of subjects that commenced the trial participated until completion of the trial. Multilevel analysis uniquely allows the use of unbalanced data sets. The variance component model using restricted maximum likelihood estimation was used in the hierarchical model building strategy. Absent data were assumed to be 'missing at random', implying that the reason for the missing data is not relevant to the phenomena being investigated [ 14 ]. Randomisation The success of randomisation was verified by dividing the subjects into groups to assess whether the distribution of group characteristics was evenly balanced. The group was divided by age, gender, BMI and gender and is given in table 1 . The randomisation process resulted in an uneven distribution of group characteristics according to occupation level and gender. The numbers and proportions of the distribution of staff by occupation level are provided in table 2 . The majority, 71%, of the staff randomised into the placebo group were administration staff, while the fish oil group was fairly evenly distributed. In addition, males seem to be under-represented in the placebo group. Table 1 Distribution of group characteristics for subjects randomised into intervention study. Treatment group Control: no-treatment Placebo: olive oil Active: fish oil Mean SD Mean SD Mean SD Age 44.18 8.127 44.43 9.387 40.69 7.391 Gender .74 .443 .79 .426 .63 .500 Body Mass Index .00 .00 28.051 9.2384 26.684 5.8819 Occupation Status .40 .494 .29 .469 .56 .512 Note: Dummy variables given to categorical variables: gender is coded 1 = female and 0 = males, and occupation status is coded 0 = administration staff and 1 = academic staff. Table 2 Distribution of staff by occupation and gender between the three treatment groups at baseline. Control Placebo Active No-treatment Olive Oil Fish Oil n % n % n % Academic 26 40 4 29 9 56 Administration 37 60 10 71 7 44 Total 63 100 14 100 16 100 Male 17 26 3 21 6 38 Female 46 74 11 79 10 63 Total 63 100 14 100 16 100 Note: % = relative proportion for each subcategory (occupation and gender), n = number of subjects These frequencies and distributions are not outside the range of expected results from the randomisation process. The main concern is that the numbers are very low in some groups. For instance, 3 male subjects in the placebo group and 4 academic staff in the placebo group may not be enough to be sensitive to significant effects, increasing the risk of a Type II error. However, the results of multivariate analysis of variance found that gender and occupational status did not have any effects on perceived stress. Therefore, the unbalanced distribution of group characteristics was not relevant. Preliminary and multilevel analysis Correlations between PSS scores at Time 1 and baseline were high (0.9, p < 0.05) and there was no significant difference between group means on paired-samples t-tests. Therefore all subsequent analysis treated Time 1 PSS as baseline scores. Preliminary analysis involved testing for any main effects of occupation level, gender and age on the various stress measures. A two-way between-groups multivariate analysis of variance was used to test for significant interactions between the active (fish oil) and placebo (olive oil) groups over time. Age, gender and occupation levels were all added to the model with no significant effects. Therefore all models were reduced to test for the two main effects of treatment and time on perceived stress. When the fish oil group was compared with the placebo group (olive oil), variance components analysis found no difference. No treatment effect was found but there was a trend towards an effect for the fish oil group (Wald statistic = 1.24). There was a main effect for time in both groups. Because of the very small numbers of subjects on the study, results of the comparison between the fish oil and the placebo groups were then compared with the group of subjects that did not participate in the intervention study. Means and standard deviations for the three groups on the PSS are given in table 3 . The means were much higher for subjects on the intervention study. A hierarchical model, given in table 4 , investigated the effects of time, treatment and the interaction of time on treatment for the three treatment groups, where T1 = no treatment, T2 = placebo (olive oil), and T3 = active (fish oil). Table 3 Means and standard deviations for the three groups on the PSS No-treatment Placebo (olive oil) Active (fish oil) Mean ( SD ) Mean ( SD ) Mean ( SD ) n 63 14 16 PSS 15.83 (4.79) 23.93 (2.62) 23.94 (4.33) Note: n = number of subjects Table 4 Parameter estimates for models of variance components, time, treatment (T1 and T2) and interaction (of time on treatment) main effects on the Perceived Stress Scale (PSS) group means. Variance Components Time Main Effects Treatment Main Effects Interaction Main Effects Fixed effects Coeff. (S.E.) Coeff. (S.E.) Coeff. (S.E.) Coeff. (S.E.) β Constant 17.639 (0.533) 18.444 (0.597) 16.479 (0.598) 15.836 (0.614) Time -1.999 (0.669)* -2.223 (0.665)* -0.500 (0.770) T2 6.958 (1.241)* 8.092 (1.441)* T3 5.372 (1.164)* 8.101 (1.365)* T2 × time -2.786 (1.679) T3 × time -6.059 (1.554)* Random effects Subject level residual variance 15.498 (4.260) 16.639 (4.200) 8.226 (3.063) 9.867 (2.956) Time level residual variance 18.496 (3.115) 16.599 (2.801) 16.721 (2.801) 13.930 (2.334) -2 log likelihood 1020.264 1011.772 974.964 960.204 * Denotes significance (Wald statistic ≥ 1.96, where p ≤ 0.05). Note: T2 = olive oil group, T3 = fish oil group, Coeff. = coefficient, S.E. = standard error, -2 log likelihood statistic = -2LL. The effects of time were significantly greater for subjects on the intervention study. There was a significant effect of treatment in the respective active and the placebo groups. Once the effects of time were accounted for, only the fish oil group estimated means were significantly different. The changes in the group means over time are provided in table 5 and illustrated in figure 3 . There were substantial changes on the PSS for the two groups in the intervention study over time, but the changes in the no-treatment group were not different over time. Table 5 Predicted changes over time in estimates of Perceived Stress Scale (PSS) means for each treatment group, 95% confidence intervals, and chi square value. Control Placebo Fish oil Estimate -0.500 -3.285 -6.559 95% CI sep 1.509 2.923 2.646 95% CI joint 2.152 4.170 3.755 Chi square 0.421 4.852* 23.590* * significance determined by chi sq ≥ 3.84, the critical value of chi square for df = 1 when p ≤0.05. Note: 95% CI sep = 95% confidence interval considering each item separately, 95% CI joint = 95% confidence interval, considering the estimates of the three treatment groups collectively, df = degrees of freedom Figure 3 Changes over time in predicted means on the Perceived Stress Scale (PSS) for the three treatment groups. The change over time in PSS means were not the same for the two groups in the intervention study. Inclusion in the fish oil group predicted a larger reduction in estimates of perceived stress than the placebo group. Estimated means at Time 2 on the PSS were 17.4 for the fish oil group, where those for the placebo group were 20.6. The within-group changes over time are given in table 6 . The changes over time were significant for both the placebo and fish oil groups but not the no-treatment group. The difference between the groups in the changes over time are given in table 7 . The differences between the fish oil group and the no-treatment group is the only between-group difference to reach statistical significance ( p < 0.05). The change over time in the placebo group was not significantly different from those in the no-treatment group. Changes in the placebo group over time were also not significantly different from changes in the fish oil group over time. Table 6 Predicted means and 95% confidence intervals of the Perceived Stress Scale (PSS) for the treatment groups over time. Control Placebo Fish oil Time 1 Mean 15.836 23.929 23.938 95% CI sep 1.203 2.555 2.390 95% CI joint 1.716 3.645 3.409 Time 2 Mean 15.337 20.643* 17.379* 95% CI sep 1.416 2.725 2.455 95% CI joint 2.021 3.888 3.502 Note: 95% CI sep = 95% confidence interval considering each item separately, 95% CI joint = 95% confidence interval, considering the estimates of the three treatment groups collectively. denotes significant difference from baseline ( p < 0.05). Table 7 Estimated between-group differences in changes over time for predicted means on the Perceived Stress Scale (PSS), with confidence intervals and chi square statistic. Placebo vs Control Fish oil vs Control Fish oil vs Placebo Estimate 2.786 6.059 3.273 95% CI sep 3.289 3.046 3.943 95% CI joint 4.692 4.345 5.625 Chi square 2.754 15.193 2.647 * Significance determined by observed value of chi sq ≥ 3.84, the critical value of chi square for df = 1 when p ≤ 0.05 Note: 95% CI sep = 95% confidence interval considering each item separately, 95% CI joint = 95% confidence interval, considering the estimates of the three treatment groups collectively, chi sq = chi square, vs = versus, the difference between groups Discussion There were significant reductions in stress for both the fish oil and the placebo (olive oil) groups from baseline (both p < 0.05). The stress reduction for the fish oil group was significantly different from the no-treatment controls ( p < 0.05). The stress reduction in the placebo group was not significantly different from the no-treatment controls. The fish oil group had more substantial stress reductions than the olive oil group, but the differences between the fish oil and the placebo groups did not reach statistical significance. All subjects taking the nutritional intervention reported significantly less perceived stress at Time 2 ( p < 0.05). Arguably, the key factor influencing these results is 'regression to the mean', an effect that may have been exaggerated by methodological limitations such as timing, a large placebo effect, and the possibility that the placebo was not a true placebo. These issues are discussed in the following sections. Statistical regression Perhaps staff applied and were selected for the intervention study at a time when their stress levels were peaking. If this were the case, 'regression to the mean' predicts that stress levels would decrease. Factors which would exaggerate this effect are the difference in the means between the groups on the intervention study and the no-treatment controls, and the study time course corresponding with the mid-year break. Because only 30 out of a possible 47 candidates were randomised into the intervention study, the 17 potential candidates remaining in the group receiving no intervention were thought to increase the mean of the no-treatment group. That is, 37% of the no-treatment group had high scores on the PSS (≥ 17). Therefore, this group was considered a potential control group for the intervention study. The time course of the study may have noticeably influenced the regression to the mean. The academic year at Southern Cross University consists of two semesters. This study commenced mid-way through the first semester (Time 1). Questionnaires were reissued at the beginning of the second semester (Time 2), immediately after the mid-year break. Staff may generally have been more relaxed and less stressed after the break. The variation of stress levels associated with time may have been minimised if Time 2 had been at mid-semester 2, a time that corresponds more closely with Time 1. Evidence of a treatment effect beyond statistical regression will be demonstrated in the between-group differences. The only such difference was between the fish oil group and the no-treatment controls. This evidence supports the hypothesis that fish oil ameliorates chronic stress. Methodological limitations The present study has several important limitations, including (1) selection bias (2) unsuccessful blinding (3) inadequate power and (4) the possibility of an active effect from the 'placebo'. Sampling bias Invariably, recruitment involving self-selection entails some degree of selection bias [ 15 ]. In this instance, the advertisement was aimed at university staff interested in stress research involving the beneficial omega-3 fatty acids. Staff may have excluded themselves for weight-watching reasons, or included themselves because they were interested in the intervention. Perhaps very stressed and/or busy staff did not apply, or perhaps staff that were not stressed did not apply. Because the study ran into the mid year break, perhaps staff did not apply because of issues relating to availability during the break. Power While the results demonstrated a strong trend towards a difference between the fish oil and the olive oil groups, this difference failed to reach statistical significance with the current sample size. Post-hoc power calculations were conducted using the PSS means and standard deviations. The study had 90% power to find a 20% difference between the fish oil and the placebo groups, and only 40% power to find a difference of 10% significant. The study was underpowered to find the difference between the groups statistically significant. Blinding Although the capsules appeared in every way identical, there was one distinct difference. The fish oil capsules were often accompanied by very mild gastrointestinal disturbances in the form of a slight after taste in the mouth, which was unmistakably fishy. All the subjects taking the fish oil suspected as much because they had the taste of fish in their mouth after ingestion of the capsules. Interestingly, half (50%) the subjects taking the placebo during this study also believed they were taking the active because they 'felt better'. The placebo group did actually report significantly less stress levels. This effect may be due regression to the mean, as previously discussed. Other factors that must be considered are the 'placebo effect' and the possibility of an active effect from the placebos. The placebo effect Half (50%) of the subjects taking the placebo believed themselves to be taking the active. If these subjects believed in the treatment and expected to benefit from it, then it is likely they reported the improvements, which may have influenced the mean of the group. If half the group were reporting an exaggerated stress reduction, then the mean of the group would show a trend towards a treatment effect. Indeed, this was observed: The placebo group demonstrated significant stress reduction; however, the change over time was not significantly different from reductions observed in the no-treatment controls. Perhaps the most unbiased way to estimate true placebo effects is to observe the difference between a placebo group and a group of no-treatment controls in a three-arm clinical study [ 16 ]. In the present study, the change over time for the placebo group, like the fish oil group, was significant ( p < 0.05), where the change over time for the no-treatment controls was not significant. This apparent placebo effect, however, may have been inflated by statistical regression or an active effect from the placebo. The placebo The use of olive oil as a placebo is not uncommon in essential fatty acid research [ 17 ]. However, olive oil may not be an inert substance in brain lipid chemistry. Oleic acid, the major lipid in olive oil, is related to an endogenous sleep-inducing substance. Isolation of a chemical from the cerebrospinal fluid of sleep deprived cats, led to its identification as cis -9,10-octadecenoamide (oleamide), an analogue of 9-octadecenoic acid (oleic acid) [ 18 ]. Oleamide, but not oleic acid, was found to improve the action of serotonin (5-hydroxytryptamine), which implies a role for this molecule in mood, alertness and sleep [ 19 ]. Cell membranes have been shown to catalyse the synthesis of oleamide from oleic acid [ 20 ]. In addition, the rat brain demonstrated control of increased levels of cis -9,10-octadecenoamide (oleamide) by conversion into oleic acid [ 18 ]. Understanding of the lipid chemistry involved with the neuromodulation of sleep and mood is still incomplete. However, the assumption that oleic acid is neutral in lipid neurochemistry is questionable. The issue of a correct choice of placebo for essential fatty acid research is difficult. Most studies use omega-6 rich oil, such as corn oil, soy oil, or safflower seed oil. However, this practice is not completely unbiased as the omega-6 oil competes with omega-3 fatty acids for metabolism. Given in high enough doses, the omega-6 oils will overwhelm delta-6 desaturase and inhibit the metabolism of the omega-3 fatty acids. This methodology results in a systematic measurement error, where the omega-3 metabolism in the placebo group may be suppressed. Further, the placebo is not a true placebo if it has a specific effect on the measurement outcome. Between-group differences in these studies may be enhanced. Alternatively, soy oil has been shown to increase omega-3 levels in omega-3 deficiency. As the regulation of the end products of n-3:n-6 blood levels is complex and not fully understood, it is probably best to avoid using these fatty acids as a placebo. This practice may increase the variance in the measurement error to an unknown extent. The monounsaturated fatty acid found in olive oil was chosen here to avoid further imbalances between the essential fatty acids. Conclusions Perceived stress was significantly reduced from baseline after 6 weeks supplementation with 1.5 g/d DHA from fish oil ( p < 0.05). Furthermore, the difference was significant compared with no-treatment controls ( p < 0.05). The placebo group also demonstrated significant reductions in perceived stress compared to baseline levels ( p < 0.05). However, when compared with the no-treatment control group, the differences in perceived stress were not significant for the placebo group. The study may have demonstrated an exaggerated regression to the mean due to its timing, a strong placebo effect or the placebo itself may have had an active effect. The question that olive oil may have a subtle but protective effect in stress has nevertheless been raised. Perhaps 6 g per day of olive oil was not sufficient dietary intake to find a difference between the placebo and no-treatment controls significant, especially with such a small sample. This research has provided preliminary findings suggesting that DHA ameliorates perceived stress. Future research is required to conclusively substantiate the ameliorating effects of DHA in stress, and further investigate the role of olive oil and/or other dietary fats in stress reduction. Competing interests This research was purely academic research and has no commercial interest. However, Blackmores Ltd, a company that sells fish oil capsules, also employs one of the authors, Chris Oliver.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC538287.xml
368172	N-Terminal Phosphorylation of the Dopamine Transporter Is Required for Amphetamine-Induced Efflux	Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA efflux into the synaptic cleft. We previously demonstrated that a human DAT construct in which the first 22 amino acids were truncated was not phosphorylated by activation of protein kinase C, in contrast to wild-type (WT) DAT, which was phosphorylated. Nonetheless, in all functions tested to date, which include uptake, inhibitor binding, oligomerization, and redistribution away from the cell surface in response to protein kinase C activation, the truncated DAT was indistinguishable from the full-length WT DAT. Here, however, we show that in HEK-293 cells stably expressing an N-terminal-truncated DAT (del-22 DAT), AMPH-induced DA efflux is reduced by approximately 80%, whether measured by superfusion of a population of cells or by amperometry combined with the patch-clamp technique in the whole cell configuration. We further demonstrate in a full-length DAT construct that simultaneous mutation of the five N-terminal serine residues to alanine (S/A) produces the same phenotype as del-22—normal uptake but dramatically impaired efflux. In contrast, simultaneous mutation of these same five serines to aspartate (S/D) to simulate phosphorylation results in normal AMPH-induced DA efflux and uptake. In the S/A background, the single mutation to Asp of residue 7 or residue 12 restored a significant fraction of WT efflux, whereas mutation to Asp of residues 2, 4, or 13 was without significant effect on efflux. We propose that phosphorylation of one or more serines in the N-terminus of human DAT, most likely Ser7 or Ser12, is essential for AMPH-induced DAT-mediated DA efflux. Quite surprisingly, N-terminal phosphorylation shifts DAT from a “reluctant” state to a “willing” state for AMPH-induced DA efflux, without affecting inward transport. These data raise the therapeutic possibility of interfering selectively with AMPH-induced DA efflux without altering physiological DA uptake.	Introduction The dopamine transporter (DAT) plays a critical role in the synaptic clearance of dopamine (DA) by mediating the reuptake of DA released into the presynaptic terminal ( Amara and Kuhar 1993 ; Giros and Caron 1993 ). It thereby regulates the strength and duration of the dopaminergic response. DAT is also the site of action of several psycho-stimulant drugs, including amphetamine (AMPH) and cocaine ( Kuhar et al. 1991 ). As a substrate, AMPH competitively inhibits DA reuptake, thereby increasing synaptic DA concentration and enhancing the rewarding property of the dopaminergic system. Additionally, AMPH elicits the release of DA through the transporter in the brain ( Fischer and Cho 1979 ; Jones et al. 1998 ) and in heterologous cells expressing DAT ( Eshleman et al. 1994 ; Wall et al. 1995 ; Sitte et al. 1998 ). AMPH-induced DA efflux is thought to be mediated by a facilitated exchange diffusion process, in which inward transport of substrates increases the availability of inward-facing binding sites of the transporter ( Fischer and Cho 1979 ), which leads thereby to increased efflux of cytosolic substrates. Emerging evidence, however, indicates that inward and outward transport of monoamines may differ in more fundamental ways. In particular, it appears that AMPH-induced DA efflux does not rely exclusively on the ability of AMPH to increase the availability of inward-facing DATs ( Chen and Justice 2000 ) but also relates to the ability of AMPH to induce uncoupled currents ( Sitte et al. 1998 ) and to increase intracellular sodium ( Khoshbouei et al. 2003 ) and kinase activity ( Kantor and Gnegy 1998 ). Although AMPH-induced currents have been shown to be of physiological relevance ( Ingram et al. 2002 ), AMPH exerts its primary behavioral effects by inducing DA efflux ( Wise and Bozarth 1987 ; Sulzer and Galli 2003 ). In addition, enhanced AMPH-induced DA efflux is associated with sensitization to repeated AMPH administration ( Robinson and Becker 1986 ). DAT is thought to comprise 12 transmembrane segments with cytoplasmic N-terminal and C-terminal domains ( Giros and Caron 1993 ). There are numerous putative phosphorylation sites for various protein kinases in the intracellular domains ( Giros and Caron 1993 ; Granas et al. 2003 ; Lin et al. 2003 ), and multiple protein kinases have been shown to regulate DAT function ( Daniels and Amara 1999 ; Melikian and Buckley 1999 ; Granas et al. 2003 ). Treatment with AMPH also leads to increased intracellular accumulation of DAT ( Saunders et al. 2000 ), and AMPH has been shown to increase striatal particulate PKC activity ( Giambalvo 1992 ) through a calcium dependent pathway ( Giambalvo 2003 ). Importantly, PKC activation leads to N-terminal phosphorylation of DAT in rat striatum ( Foster et al. 2002 ). Consistent with this observation, we recently showed that deletion of the first 22 amino acids from DAT essentially eliminates 32 P incorporation into DAT in response to PKC activation ( Granas et al. 2003 ). Surprisingly, this truncation did not affect PKC-induced internalization, thereby demonstrating that N-terminal phosphorylation of DAT is not essential for internalization. Since uptake, inhibitor binding, and oligomerization of this truncated DAT were also not significantly different from those of full-length DAT ( Hastrup et al. 2001 , 2003 ; Granas et al. 2003 ), N-terminal phosphorylation has not yet been associated with a functional effect. PKC activation, however, has been shown to stimulate DAT-mediated release of DA ( Davis and Patrick 1990 ; Giambalvo 1992 ; Kantor and Gnegy 1998 ). Moreover, AMPH-induced DA efflux is inhibited by the introduction of PKC inhibitors and by downregulation of PKC ( Kantor and Gnegy 1998 ; Cowell et al. 2000 ; Kantor et al. 2001 ), whereas DA uptake is unaffected by these manipulations. This suggests that inward and outward transport can be independently regulated and led us to explore the hypothesis that N-terminal phosphorylation of DAT may be involved in AMPH-induced DA efflux. Here we report that deletion of the first 22 amino acids of DAT, as well as mutation of the five N-terminal serines to alanine, greatly decreases AMPH-induced DA efflux without affecting uptake. Mutation of these serines instead to aspartate, thereby mimicking phosphorylation, preserves efflux, suggesting that phosphorylation of one or more of these five N-terminal serines is essential for AMPH-induced DA release. Results/Discussion In our previous studies we created a mutant human DAT construct in which the first 22 amino acids were removed and replaced by tandem FLAG and HA epitope tags (FLAG-HA-DAT) ( Hastrup et al. 2001 , 2003 ). This construct was created to tag the protein and to remove Cys6 to facilitate biochemical studies. FLAG-HA-DAT expressed at wild-type (WT) levels in the plasma membrane, and we found it to be functionally normal in terms of uptake, inhibitor binding, DAT oligomerization, and PMA- and receptor-induced internalization FLAG-HA-DAT expressed at wilde-type (WT) levels in the plasma membrane, and we found it to be functionally normal in terms of uptake, inhibitor binding, DAT oligomerization, and PMA- and receptor-induced internalization (Hastrup et al. 2001, 2003; Granas et al. 2003). Since this construct lacks the first five serines in DAT (Ser2, Ser4, Ser7, Ser12, Ser13) and does not appear to be phosphorylated by PKC activation ( Granas et al. 2003 ), we hypothesized that FLAG-HA-DAT might be impaired in AMPH-induced efflux. In accordance with this prediction, we found that AMPH-induced DA efflux was decreased by approximately 80% in the FLAG-HA construct relative to FLAG-tagged full-length DAT (FLAG-DAT) ( Figure 1 ). This resulted from a decrease in the maximal rate of DA efflux and not from a change in the apparent affinity for AMPH in mediating efflux. In contrast, DA uptake by these two constructs was not significantly different ( Figure 1 , legend). Figure 1 N-Terminal Truncation of DAT Impairs AMPH-Induced DA Efflux Cells were preloaded with 15 μM DA and superfused with AMPH at concentrations ranging from 1 to 100 μM. AMPH-induced DA efflux was defined as the amount of DA released in response to the given concentration of AMPH minus the baseline value. Baseline DA release did not differ between FLAG-HA-DAT and FLAG-DAT (13.2 ± 2.9 and 10.2 ± 1.8, respectively; n = 18). The V max of efflux was 31.1 ± 4.6 and 128.3 ± 12.0 pmol/mg protein/fraction (F(2,27) = 52.6, p < 0.0001) with a K m for amphetamine of 7.8 ± 4.1 and 7.6 ± 2.2 μM, for FLAG-HA-DAT and FLAG-DAT, respectively ( n = 4). For [ 3 H]DA uptake, the V max was 15.4 ± 2.5 and 18.3 ± 2.2 pmol/min/mg protein with a K m of 1.2 ± 0.8 and 1.1 ± 0.4 μM for FLAG-HA-DAT and FLAG-DAT, respectively (F(2,49) = 1.78, p > 0.17). In a cell suspension (or in a population of adherent cells), it is difficult to assess the potential effects on efflux of a change in ionic gradients or membrane potential because the membrane potential and ionic gradients change freely depending on the stimuli. Indeed, AMPH has been shown to induce depolarization through a DAT-mediated uncoupled chloride conductance that can be gated by substrates such as AMPH ( Ingram et al. 2002 ). Therefore, in order to quantify these effects under conditions where we could control the intracellular concentration of the substrates, DA, sodium, and chloride, as well as the membrane potential, we used amperometry in conjunction with the patch-clamp technique in the whole-cell configuration, a method that we have used previously to study the mechanism of efflux ( Galli et al. 1998 ; Khoshbouei et al. 2003 ). We recorded DAT-mediated currents with the whole-cell pipette by stepping the membrane voltage from a holding potential of −20 mV to +100 mV while simultaneously measuring efflux as assessed by amperometric currents resulting from the release of DA. Consistent with our studies with cell populations, we found that AMPH-induced efflux was decreased at +100 mV by 91% ± 4% ( n = 5) in FLAG-HA-DAT relative to FLAG-DAT. Surprisingly, the DAT-mediated whole-cell currents gated by AMPH, which have been shown to be uncoupled from the transport process ( Sonders et al. 1997 ; Khoshbouei et al. 2003 ), were also reduced to a comparable extent (see below). This reduction in current and efflux resulted from the N-terminal deletion and not from the presence of the HA epitope, since a FLAG-tagged construct lacking the first 22 amino acids of DAT (FLAG-del22-DAT) but without any other added sequence showed a reduction in current and efflux similar to that of FLAG-HA-DAT. Figure 2 shows representative traces for the AMPH-induced current and DA efflux recorded at +100 mV obtained from FLAG-DAT (panels A and B, respectively) and FLAG-del22-DAT (panels C and D, respectively). In panels B and D, the upward (positive) deflections indicate DA oxidation and thus reflect DA efflux. At the onset of the voltage step, the amperometric electrode recorded an oxidative current (positive), which is indicative of DA efflux, and at the termination of the voltage step, the amperometric current relaxed to baseline. At +100 mV, the AMPH-induced whole-cell and oxidative currents recorded from FLAG-del22-DAT cells were much smaller than those recorded from FLAG-DAT cells: in FLAG-del22-DAT cells, the whole-cell currents were 21.8% ± 7.4% whereas the amperometric currents were 23.0% ± 2.5% of the equivalent currents recorded in FLAG-DAT cells ( n = 5). Figure 2 N-Terminal Truncation of DAT Reduces AMPH-Induced Currents and DA Efflux Cells were voltage clamped with a whole-cell patch pipette while an amperometric electrode was placed onto the cell membrane. The internal solution of the whole-cell patch pipette contained 2 mM DA. (A) Representative trace of AMPH-induced whole-cell current obtained from FLAG-DAT cells upon AMPH (10 μM) bath application. The membrane potential of the cell was stepped to +100mV from a holding potential of –20 mV. (B) Oxidation current acquired concomitantly to the whole-cell current represented in panel A. (C and D) Representative current traces (whole-cell and amperometric, respectively) obtained from FLAG-del22-DAT cells using the same experimental protocol as in (A) and (B). In marked contrast to this approximately 80% reduction, in the same two sets of stably transfected cells, the V max for uptake of the substrate tyramine by FLAG-del22-DAT was 146% that by FLAG-DAT ( Table 1 ). Neither the K m for tyramine uptake ( Table 1 ) nor the apparent K i for inhibition of tyramine uptake by AMPH (37 ± 4 nM and 63 ± 18 nM, respectively; n = 5) or cocaine (214 ± 34 nM and 281 ± 33 nM, respectively; n = 4) was significantly different in FLAG-DAT and FLAG-del22-DAT. Cell-surface biotinylation studies revealed that the increased V max in FLAG-del22-DAT was accounted for by an increased number of DAT molecules at the cell surface ( Table 1 ) and suggested that the truncation had a minimal effect on the turnover rate of the transporter. These results are consistent with our previous studies on the FLAG-HA-DAT deletion construct expressed in EM4 cells, which also showed normal tyramine uptake ( Hastrup et al. 2001 ), as well as with the DA uptake studies described above for FLAG-HA-DAT and FLAG-DAT expressed in HEK-293 cells (see Figure 1 , legend). Table 1 Kinetic Properties of [ 3 H]Tyramine Uptake and Cell-Surface Localization of FLAG-DAT, FLAG-del22-DAT, FLAG-S/A-DAT, and FLAG-S/D-DAT a The surface biotinylation data (mean ± SEM, n = 5) are arbitrary units obtained from the analysis of immunoblots. Since FLAG-DAT and FLAG-del22-DAT were studied in parallel, and FLAG-S/D-DAT and FLAG-S/A-DAT were studied in parallel, the data for each set were normalized to the construct with normal efflux, and therefore these values are shown as 1. Uptake data represent mean ± SEM of 5–6 experiments If the reduction in the AMPH-induced current and efflux resulted from the loss of phosphorylation of one or more of the five N-terminal serine residues, then mutation of the serine(s) that is (are) phosphorylated should lead to an effect similar to that of the truncation. Since it is not known which of the serines are phosphorylated, we simultaneously mutated all five serines to alanine in the full-length FLAG construct (FLAG-S/A-DAT). To obtain further evidence that phosphorylation of one or more of the N-terminal serines is essential for AMPH-induced DA efflux, we also created a construct in which all five of these serines were simultaneously mutated to aspartate (FLAG-S/D-DAT), in an attempt to simulate phosphorylation of the serines. Neither the K m nor the V max for tyramine uptake was significantly different in FLAG-S/A-DAT and FLAG-S/D-DAT (see Table 1 ). The small, nonsignificant reduction in uptake by FLAG-S/A-DAT was accounted for by a similarly decreased number of DAT molecules at the cell surface (see Table 1 ), suggesting that the turnover rate of the transporter was the same in these two mutants. The apparent K i 's for inhibition of tyramine uptake in FLAG-S/A-DAT and FLAG-S/D-DAT by AMPH (41 ± 13 nM and 48 ± 7 nM, respectively; n = 3) or by cocaine (331 ± 46 nM and 444 ± 47 nM, respectively; n = 4) were not significantly different. Current-voltage and amperometric-voltage relationships were generated for FLAG-DAT, FLAG-del22-DAT, FLAG-S/A-DAT, and FLAG-S/D-DAT by stepping the voltage from a holding potential of −20 mV to voltages between –120 mV and +100 mV in increments of 20 mV ( Figure 3 ). In FLAG-DAT cells, AMPH-induced currents and DA efflux were voltage dependent, with an increase at positive voltages and saturation of DA efflux near +100 mV ( Figure 3 A and 3 B, filled circles). In contrast, in FLAG-del22-DAT cells, the AMPH-induced currents and DA efflux were greatly reduced at all voltages tested (compare Figure 3 A and 3 B, open circles, with Figure 3 A and 3 B, filled circles). This phenomenon was not likely a consequence of an alteration of ion gradients or accumulation of intracellular AMPH, because no significant differences were found between the reversal potentials of the current obtained from FLAG-DAT cells (24.5 ± 5.3 mV) and FLAG-del22-DAT cells (32.6 ± 6.3 mV). In FLAG-DAT cells, the amperometric current at +80 mV was 0.305 ± 0.079 pA (mean ± SEM; n = 6) ( Figure 3 B, filled circles). In contrast, in FLAG-del22-DAT cells the amperometric current recorded at the same potential was significantly reduced (0.077 ± 0.028 pA, mean ± SEM; p < 0.05 by Student's t-test, FLAG-del22-DAT versus FLAG-DAT; n = 5) ( Figure 3 B, open circles). Figure 3 AMPH-Induced Current-Voltage and Amperometric-Voltage Relationships Obtained from FLAG-DAT, FLAG-del22-DAT, FLAG-S/A-DAT, and FLAG-S/D-DAT (A) Current-voltage relationships of AMPH-induced current obtained from FLAG-DAT (filled circles) and FLAG-del22-DAT (open circles) cells. AMPH (10 μM) was applied to the bath while the membrane potential was stepped from–120 mV to +100 mV from a holding potential of –20 mV in 20 mV increments ( n = 5). (B) Amperometric-voltage relationships obtained from FLAG-DAT (filled circles) and FLAG-del22-DAT (open circles) cells acquired concomitantly to the whole-cell current of panel A. (C and D) Current-voltage (C) and amperometric-voltage (D) relationships of whole-cell and oxidative currents obtained from FLAG-S/D-DAT (filled triangles) and FLAG-S/A-DAT (open triangles) cells using the same experimental protocol as above. Similarly, in FLAG-S/D-DAT cells the AMPH-induced currents and DA efflux were much greater than those generated in FLAG-S/A-DAT cells ( Figure 3 C and 3 D, filled triangles and open triangles, respectively). In FLAG-S/D-DAT cells, the amperometric current at +80 mV was 0.202 ± 0.039 pA (mean ± SEM; n = 7) ( Figure 3 D, filled triangles). In contrast, in FLAG-S/A-DAT cells, the amperometric current recorded at the same potential was significantly reduced (0.014 ± 0.009 pA, mean ± SEM; p < 0.05 by Student's t-test, FLAG-S/D-DAT versus FLAG-S/A-DAT; n = 5). Thus, the ability of AMPH to induce DAT-mediated currents and DA efflux was impaired dramatically, either by N-terminal truncation, or by substitution of the five N-terminal serines to alanine. Remarkably, substituting these five serines to aspartate to mimic phosphorylation restored the ability of AMPH to induce voltage-dependent DA efflux and to produce currents, indicating that negative charges in the DAT N-terminal region are essential for these actions of AMPH. To explore which serine or serines are critical to the effect on efflux, we created five additional mutants in the FLAG-S/A-DAT background in which we mutated each of the five positions, one at time, to aspartate, and we created stable pools of EM4 cells expressing each of these mutants. At +100 mV the amperometric currents in FLAG-S/A-DAT, FLAG-S/A-2D-DAT, FLAG-S/A-4D-DAT, and FLAG-S/A-13D-DAT were 7.4% ± 2.6%, 8.4% ± 5.7%, 11.2% ± 3.1%, and 12.3% ± 7.0%, respectively, of that seen in FLAG-S/D-DAT ( n = 3; not significantly different from FLAG-S/A-DAT by One-way ANOVA and Tukey's Multiple Comparison Test). In contrast, amperometric currents in FLAG-S/A-7D-DAT and FLAG-S/A-12D-DAT were 29.8% ± 12.6% and 45.1% ± 9.6%, respectively, of that seen in FLAG-S/D-DAT ( n = 3; p < 0.01 compared to FLAG-S/A-DAT by One-way ANOVA and Tukey's Multiple Comparison Test). Thus, negative charge at either position 7 or position 12 restores a substantial fraction of the efflux seen with aspartate at all five positions, and the size of the resulting efflux relative to FLAG-S/D-DAT and FLAG-DAT suggests that both of these serines may be phosphorylated in vivo (see below). The differences in AMPH-induced DA efflux between FLAG-S/A-DAT and FLAG-S/D-DAT could result either from an altered affinity of DAT for intracellular DA or from a change in the V max of the transport process. At +80 mV, at what is a saturating concentration of intracellular Na + for FLAG-DAT (see “Materials and Methods” ), the K m for intracellular DA was 1.4 ± 0.4 mM for FLAG-S/A-DAT and 1.3 ± 0.4 mM for FLAG-S/D-DAT. Thus, a change in the V max of the AMPH-induced DAT-mediated efflux is likely responsible for the differences between FLAG-S/A-DAT and FLAG-S/D-DAT. Our results suggest that phosphorylation of one or more serines in the N-terminus of the human DAT shifts DAT from a “reluctant” state to a “willing” state for AMPH-induced DA efflux. (A related phenomenon has been proposed for calcium channel regulation [ Zhu and Ikeda 1994 ].) That DAT is significantly phosphorylated under basal conditions and that this phosphorylation can be increased by AMPH (Roxanne Vaughan, pers. comm.) are also consistent with a role for N-terminal phosphorylation in the AMPH-induced efflux mechanism. The structural basis for this regulation of efflux is currently unknown. It may result from a shift in the voltage or sodium dependence of efflux and thus from an increase in the fraction of DAT molecules that reorient to the external milieu empty of DA. Whatever the mechanism, under unclamped, “physiological” conditions, N-terminal phosphorylation does not alter significantly any rate-limiting steps for uptake. Despite our demonstration that the V max for uptake is unaltered in the mutants, it is possible that phosphorylation might alter the ionic coupling of DAT. The ratio of whole-cell to amperometric current ( Galli et al. 1997 ) at +100 mV was not different in FLAG-DAT and FLAG-del22-DAT (728 ±193 [ n = 8] and 835 ± 300 [ n = 5], respectively; p > 0.05 by Student's t test). (Similar results were obtained at +60 and +80 mV [data not shown].) This ratio is a microscopic property of an individual transporter that is inversely proportional to the fraction of charge carried by dopamine ( Galli et al. 1997 ). These data, measured in the presence of saturating intracellular dopamine concentrations in the patch pipette, are consistent, therefore, with a similar ionic coupling in the two mutants. However, given the lack of stoichiometric coupling between substrate flux and charge movement (see below), we cannot absolutely rule out an effect of phosphorylation on the ionic coupling of flux. To rule out such a change, it would be helpful to demonstrate that the WT and mutant transporters can generate similar concentration gradients at equilibrium, even though efflux rates differ. In unclamped cells, however, the persistent presence of substrate might lead to changes in membrane potential, and, therefore, such experiments would best be performed under voltage-clamp conditions with an amperometric electrode inside the cell to measure the accumulation of dopamine ( Mosharov et al. 2003 ). Curiously, AMPH-induced currents, which are largely an uncoupled chloride conductance mediated by DAT that is gated by substrates such as AMPH ( Ingram et al. 2002 ), were reduced in the absence of N-terminal phosphorylation in parallel with DA efflux. Although the underlying mechanisms are unclear, these findings are consistent with the findings of Sitte et al. (1998 ) that there is a poor correlation between substrate-induced efflux and the uptake of substrates, but a good correlation between the ability of substrates to induce currents and their ability to cause efflux ( Khoshbouei et al. 2003 ). Regardless of the mechanisms, our findings argue that the mechanism of DA efflux is to some extent independent from the inward-transport process. Since truncation of the N-terminus had the same functional effect as neutralization of the N-terminal serines, it is likely that an essential interaction of the phosphorylated N-terminus of DAT must occur to permit efflux, either with another part of DAT or conceivably with an associated protein. These results could lead to the design and synthesis of new therapeutic agents, such as a drug that blocks the effects of AMPH-like psychostimulants without inhibiting DA uptake. Selective enhancement of DA release might be achieved by promoting phosphorylation of the N-terminus of DAT or by modulating critical interactions of the DAT N-terminus. Furthermore, a polymorphism or naturally occurring mutation of the N-terminal portion of DAT could alter efflux in the context of normal uptake, and this might be associated with human psychiatric or neurologic dysfunction, much as a polymorphism of the norepinephrine transporter has been found to be associated with orthostatic intolerance ( Robertson et al. 2001 ). Mutations of Ser7 and Ser12 of DAT were found previously to affect the response to inhibition of PKC and MEK1/2, respectively ( Lin et al. 2003 ). We found that negative charge at either of these positions, but not at the positions of the three other N-terminal serines at positions 2, 4, and 13, restored significant AMPH-induced DA efflux. Nonetheless, the serines that are actually phosphorylated as a result of activation of PKC or by AMPH have not been identified, and the kinase or kinases that directly phosphorylate the N-terminus of DAT are unknown as well. Efforts are underway to identify directly the serines that are phosphorylated in vivo, as well as the responsible kinase, and to further uncover the mechanism by which the phosphorylated N-terminus makes DAT “willing” to efflux DA. Materials and Methods Plasmid construction, transfection, and cell culture The N-terminally FLAG-tagged full-length synthetic human DAT (synDAT) gene in pCIHyg was described previously ( Saunders et al. 2000 ). In the FLAG-HA-DAT construct, an HA tag followed the FLAG tag and the first 22 amino acids (MSKSKCSVGLMSSVVAPAKEPN) of human DAT were deleted ( Hastrup et al. 2001 ). In FLAG-del22-DAT, these 22 amino acids were deleted from the full-length FLAG-DAT, making this construct identical to FLAG-HA-DAT except for the absence of the HA-tag sequence. From the FLAG-DAT background, Ser2, Ser4, Ser7, Ser12, and Ser13 were simultaneously mutated to alanine to create the FLAG-S/A-DAT construct and to aspartate to create the FLAG-S/D-DAT construct. The mutant constructs were generated, confirmed, and expressed stably in human embryonic kidney cells (HEK-293) or EM4 cells, HEK-293 cells stably transfected with macrophage scavenger receptor to promote adherence ( Robbins and Horlick 1998 ), as described previously ( Hastrup et al. 2001 ). Uptake of [ 3 H]tyramine Uptake assays with adherent EM4 cells stably expressing the appropriate DAT construct were performed as described previously ( Hastrup et al. 2001 ). Tyramine was used as a radiolabeled substrate because it is not a substrate for catechol-O-methyl transferase, which is endogenously present in HEK-293 cells and EM4 cells, and therefore is not subject to degradation that might complicate the kinetics of uptake ( Hastrup et al. 2001 ). Nonspecific uptake was determined in the presence of 2 mM tyramine. For determination of V max and K m values, increasing concentrations of tyramine from 0.02 to 50 μM were used. K m and V max values for [ 3 H]tyramine and [ 3 H]dopamine uptake were determined by nonlinear regression analysis using GraphPad Prism 4. IC 50 values were determined using increasing concentrations of AMPH between 0.002 and 2 μM and of cocaine between 0.001 and 10 μM in competition with approximately 60 nM [ 3 H]tyramine. K i values were calculated from the IC 50 values as described by Cheng and Prusoff (1973 ). Cell-surface biotinylation and immunoblotting EM4 cells stably expressing the DAT constructs were incubated with cleavable sulfo-NHS-S-S-biotin (Pierce Chemical Company, Rockford, Illinois, United States) to label surface-localized transporter, and the biotinylated material was prepared and immunoblotted as described previously ( Saunders et al. 2000 ). AMPH-induced DA efflux Confluent 100-mm plates of HEK-293 cells stably expressing FLAG-DAT or FLAG-HA-DAT were washed twice with KRH (25 mM HEPES [pH 7.4], 125 mM NaCl, 4.8 mM KCl, 1.2 mM KH 2 PO 4 , 1.3 mM CaCl 2 , 1.2 mM MgSO 4, and 5.6 mM glucose) and incubated at 37 o C with 15 μM DA for 30 min. Following incubation, cells were washed with KRH, harvested, resuspended in 0.20 ml of KRH and superfused in a Brandel superfusion apparatus (Brandel SF-12, Gaithersburg, Maryland, United States) as described by Kantor et al. (2001 ). The KRH contained 10 μM pargyline, and AMPH was added at concentrations from 1 to 100 μM for 2.5 min only. DA was determined by HPLC with electrochemical detection as described by Kantor et al. (2001 ). Electrophysiology and amperometry Whole-cell and amperometric currents were recorded as described previously ( Khoshbouei et al. 2003 ). The AMPH-induced whole-cell and amperometric currents were defined as the current recorded in the presence of AMPH, minus the current recorded after the addition of cocaine to the bath with AMPH still present. Previously, we demonstrated that AMPH increases intracellular sodium and that a high concentration of NaCl in the recording pipette maximizes DA efflux ( Khoshbouei et al. 2003 ). Thus, to increase the basal and AMPH-induced DA efflux and to maintain a constant sodium concentration, the whole-cell electrode was filled with internal solution containing 2 mM DA and 90 mM NaCl substituted with KCl to maintain a constant osmolarity of 270 mOsm. The dependence of DA efflux on internal DA was determined by fitting the values of the steady-state amperometric currents, recorded at different intracellular DA concentrations (between 500 μM and 4 mM), to a Hill equation by nonlinear regression. The ratio of whole-cell to amperometric current was calculated by dividing the average whole-cell current during the last 100 ms of the voltage step by the average amperometric current during the same time period ( Galli et al. 1998 ). Supporting Information Accession Numbers The Swiss-Prot ( http://ca.expasy.org/cgi-bin/niceprot.pl?Q01959 ) entry name for the gene discussed in this paper is S6A3_HUMAN, accession number Q01959.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC368172.xml
368166	Evolution for the Next Generation	Do you want to know about evolution? Brian and Deborah Charlesworth provide an excellent and concise account of the core issues for a broad range of readers	Evolution is a complex phenomenon that requires a broad understanding of many areas of biology for us to appreciate it fully. Moreover, the field has expanded rapidly, especially since the development of molecular techniques in the past two to three decades. Futuyma's classic text on evolution (1998) contains 26 chapters totaling 763 pages. To cover the topic in only eight chapters and 145 pages, as the Charlesworths have done in Evolution: A Very Short Introduction , is no mean feat. Their book is one of a series of short introductions, published by Oxford University Press, covering an eclectic array of subjects that aim to provide an accessible yet stimulating read for anyone wanting a thorough introduction to a topic. In this small volume, the Charlesworths have succeeded on both fronts and provide an excellent account of the core issues for a broad range of readers. One of the reasons for the book's appeal is that the authors draw on a range of carefully chosen human traits to illustrate their points. By contrast, most evolutionary textbooks (other than those purely on human evolution) tend to focus on nonhuman organisms. As with traits in every other organism, many human and human-related characteristics have evolved via genetic drift and natural selection, and they provide an effective means of convincing readers of the reality and relevance of evolution. For example, to explain how mutation can cause the loss of a function, the authors discuss the relatively poor sense of smell in humans, as compared with many other mammals, using an example of a vestigial ‘pseudogene’ of a human olfactory receptor gene. They also discuss tooth decay, enzyme aesthetics, heritable differences, cancer and other diseases, and the ability to taste and so on. Although the topics the Charlesworths choose to focus on are certainly appropriate, they provide only a brief mention of one important process—development. Evolutionary developmental biology is a burgeoning field that can provide interesting and important insights into our understanding of the mechanisms of evolution. For example, the absence of eyes in cavefish, rather than being the result of a degenerative process, might be the result of selection on genes that govern feeding morphology, a selection process that has included suppression of eye development ( Pennisi 2002 ). Such developmental mechanisms and constraints can actually alter the direction of evolution. Although the key forces driving evolution are usually thought of as mutation, genetic drift, natural selection, and divergence, the developmental pathways from genes to phenotypes, along with associated developmental constraints, can also determine the rate and direction of evolution. In Chapter 7, the authors discuss five topics that have traditionally been hard to understand from an evolutionary point of view. These ‘difficult problems’ are ageing, altruism, human consciousness, complex adaptations, and the origin of living cells. Difficult problems can be interpreted in two ways: those that, although hard to solve, have either been explained or will eventually be explained by modern evolutionary theories, and those problems that cannot be fully resolved with our current understanding but leave room for learning about additional mechanisms or factors. The Charlesworths generally consider only those problems of the former type—the explained ones. However, I think that some of the more intractable problems should be described in more detail. For instance, complex adaptation might be fully explained by mutations and natural selection, but additional unknown mechanisms might be essential for the evolution of the complex traits. I realize that opponents of modern evolutionary theory, such as creationists, have often cited these traditional problems to support their conclusion that modern evolutionary theory is wrong; but progress always depends on the consideration of new ideas, and there might be important mechanisms still to be discovered that play a key role in evolution. Describing potentially intractable problems might also spur on young readers who are thinking of studying evolutionary biology with the hope that there are still some theoretical battles to be conquered. Who is the target audience of this book? For many books, the topics chosen and the writing style can perhaps provide clues to the nature of the readers. For instance, The Blind Watchmaker by Richard Dawkins (1990) is a good introductory book for those interested in natural selection because it seems to be written mainly for individuals who either oppose or do not understand the role of natural selection. In the Charlesworths' book, providing evidence for evolution occupies 49 of the 130 pages. They explain how the similarities between living creatures can be understood in terms of evolution (Chapter 3) and subsequently discuss evidence from the geographical distributions of living and fossil species (Chapter 4). My first impression was that this part occupies too large a proportion of the book. However, Chapter 3 serves as a good introduction to the basic background of biology, such as the gene, DNA, and cells. When I read a recent article about a teaching controversy concerning evolution ( Scott and Branch 2003 ), I began to appreciate the importance—at least in the United Kingdom and the United States—of convincing readers of the reality and cogency of evolution and evolution theory by astutely providing them with the evidence to judge for themselves. In Japan, there seem to be few people who deny the facts of evolution, although there are many ideologically motivated books opposing natural selection and Darwinism. To convince creationists of evolution is usually extremely difficult, if not impossible, because they will never doubt their assumption that God created humankind. Education of young and curious people, however, can make a difference. This is where I think the book will be most successful, but this book should not just be limited to young people—I can recommend it to anyone who wants to know about evolution. Moreover, I can recommend it to Japanese students not only as an introduction to evolution, but also as an exercise in reading a well-written and engaging English text.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC368166.xml
406396	A Role for Early Cardiac Function in Cardiac Morphogenesis	xx	The heart starts beating and pumps blood through the body long before it has achieved its mature architecture. In theory, this provides a chance for cardiac function to sculpt cardiac structure, an intriguing possibility for developmental biologists, and one of potentially great clinical import for cardiologists seeking to identify the causes of (often fatal) cardiac anomalies. In this issue of PLoS Biology, Thomas Bartman et al. use the powerful tools afforded by zebrafish genetics to dissect the early steps of heart valve formation. In the process, they provide evidence for a causal relationship between the early function of the heart and its final structure. tie2::GFP+ cells form the endocardial cushions At the time of its first beat, the vertebrate heart is little more than a tube, lined on its outside by a myocardial cell layer whose contractions (the heartbeats) power blood flow, and on its inside by an endocardial cell layer, an extension of the inner wall of the connecting blood vessels. What it lacks still are valves and septae, the fibrous gates that subdivide the mature heart into atrial and ventricular chambers, and control the directionality of blood flow. These structures derive from the endocardium in a process that begins—shortly after the establishment of blood flow—with the local accumulation of endocardial cells into what are known as endocardial cushions (ECs). The zebrafish lends itself well to large-scale genetic screens, and powerful genomic tools are now available to efficiently identify the gene affected by any mutation. The authors have used genetic screens to identify several mutations that affect early cardiac function or morphology. Heart anomalies are easy to detect in zebrafish, and can be examined in real time and in live specimens because the embryos develop outside the mother and are fully transparent. Using a fluorescent molecular marker highly expressed in the ECs, the authors narrowed in on mutations that result in valve defects, and identified a mutant they named cardiofunk (cfk), which was devoid of ECs. Genetic mapping of the cfk mutation revealed a single sequence change in a gene encoding a novel actin molecule that is most closely related to the sarcomeric actins found in sarcomeres, the contracting organelles of muscle cells. The result was surprising because contractions are not a property of endocardial cells. Using RNA detection assays, the authors show that the cfk gene is in fact expressed in the myocardium, rather than in the endocardium. It therefore appears that the inability to form ECs in cfk mutants does not reside in the endocardium per se, but is an indirect consequence of a myocardial anomaly. The cfk mutation introduces a single amino acid change in the actin protein, and through detailed biochemical analyses, the authors show that the mutant actin is impaired in its ability to assemble into fibers in vitro. What might be the consequence in vivo? The authors note that cfk mutants display abnormal heart contractions prior to the development of their EC defect. Support for the notion that myocardial contractions are required for EC formation comes from the examination of silent-heart (sih) mutants. sih mutants, which lack a heatbeat, have been shown to harbor a mutation in troponin T, one of the motors of actin contractions; the authors find that sih mutants also fail to develop ECs. The mechanisms linking myocardial contractions and cushion formation remain unclear. Blood flow may be a trigger, though the authors find that ECs can develop even in the presence of pharmacological compounds that abolish it. The characterization of additional mutants should help answer this question. Valve or septal defects represent 40% of cardiac anomalies in humans. Bartman and colleagues suggest that, by analogy with zebrafish, some may result from congenital defects affecting very early myocardial function. Their work thus opens new avenues for the early detection of human cardiac malfunctions and malformations.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC406396.xml
521729	Hearing: Travelling Wave or Resonance?	A fresh look at classic work of Thomas Gold on how the inner ear processes sound	Sitting in the enveloping quietness of an anechoic chamber, or other quiet spot, you soon become aware that the ear makes its own distinctive sounds. Whistling, buzzing, hissing, perhaps a chiming chorus of many tones—such continuous sounds seem remarkably nonbiological to my perception, more in the realm of the electronic. Even more remarkable, put a sensitive microphone in the ear canal and you will usually pick up an objective counterpart of that subjective experience. Now known in auditory science as spontaneous otoacoustic emission, the sound registered by the microphone is a clear message that the cochlea uses active processes to detect the phenomenally faint sounds—measured in micropascals—that our ears routinely hear. If the ear were more sensitive, we would need to contend with the sound of air molecules raining upon our eardrums. What is that process—the mechanical or electrical scheme that Hallowell Davis in 1983 called the ‘cochlear amplifier’ ( Davis 1983 )—which energises the pea-sized hearing organ buried in the solid bone of our skull? That question has engaged my curiosity since the late 1970s, when English auditory physicist David Kemp first put a microphone to an ear and discovered the telltale sounds of the cochlea at work ( Kemp 1978 ). Siren-like, the sounds have drawn me into the theory and experiment of cochlear mechanics, now as part of a PhD course at the Australian National University in Canberra. I am studying the micromechanics of this process from a theoretical point of view, and investigating whether a resonance picture of some kind can be applied to the faint but mysterious sounds most cochleas emit. Kemp's discoveries are rightly viewed as opening a fresh path to auditory science, and to the tools and techniques for diagnosing the functional status of the cochlea. But in terms of fundamental understanding, a key paper remains that of Thomas Gold more than half a century ago ( Gold 1948 ). Still cited widely today, this paper deals with the basic question of how the cochlea works to analyse sound into its component frequencies. Two prominent theories—sympathetic resonance, proposed by Hermann Helmholtz (1885) , and travelling waves, proposed by Georg von Békésy (1960) —need to be distinguished ( Figure 1 ). In a nutshell, are there tiny, independently tuned elements in the cochlea, like the discrete strings of a piano, that are set into sympathetic vibration by incoming sound (Helmholtz), or is the continuously graded sensing surface of the cochlea hydrodynamically coupled so that, like flicking a rope, motion of the eardrum and middle ear bones causes a travelling wave to sweep from one end towards the other (von Békésy)? Figure 1 Two Views of Cochlear Mechanics The cochlea, shown uncoiled, is filled with liquid. In the accepted travelling wave picture (A), the partition vibrates up and down like a flicked rope, and a wave of displacement sweeps from base (high frequencies) to apex (low frequencies). Where the wave broadly peaks depends on frequency. An alternative resonance view (B) is that independent elements on the partition can vibrate side to side in sympathy with incoming sound. It remains open whether the resonant elements are set off by a travelling wave (giving a hybrid picture) or directly by sound pressure in the liquid (resonance alone). The first option, sympathetic resonance, has the advantage of allowing vanishingly small energies to build up, cycle by cycle, into an appreciable motion—like boosting a child on a swing. The second, travelling wave, has the weight of von Békésy's extensive experiments behind it. At the same time, one of the drawbacks of the travelling wave theory is the difficulty of accounting for the ear's exquisite fine tuning: trained musicians can easily detect tuning differences of less than 0.2%. Even von Békésy himself notes, on page 404 of his classic book, that ‘the resonance theory of hearing is probably the most elegant of all theories of hearing’. Gold's work, done in collaboration with RJ Pumphrey ( Gold and Pumphrey 1948 ), was the first to consider that the ear cannot act passively, as both Helmholtz and von Békésy had thought, but must be an active detector. Gold was a physicist who had done wartime work on radar, and he brought his signal-processing knowledge to bear on how the cochlea works. He knew that, to preserve signal-to-noise ratio, a signal had to be amplified before the detector, and that ‘surely nature can't be as stupid as to go and put a nerve fibre—that is a detector—right at the front end of the sensitivity of the system’. He therefore proposed that the ear operated like a regenerative receiver, much like some radio receivers of the time that used positive feedback to amplify a signal before it was detected. Regenerative receivers were simple—one could be built with a single vacuum tube—and they provided high sensitivity and narrow bandwidth. A drawback, however, was that, if provoked, the circuit could ‘take off’, producing an unwanted whistle. Gold connected this with the perception of ringing in the ear (tinnitus), and daringly suggested that if a microphone were put next to the ear, a corresponding sound might be picked up. He experimented, placing a microphone in his ear after inducing temporary tinnitus with overly loud sound. The technology wasn't up to the job—in 1948 microphones weren't sensitive enough—and the experiment, sadly, failed. Gold's pioneering work is now acknowledged to be a harbinger of Kemp's discoveries. But there is one aspect of Gold's paper that is not so widely considered: Gold's experiments led him to favour a resonance theory of hearing. In fact, the abstract of his 1948 paper declares that ‘previous theories of hearing are considered, and it is shown that only the resonance theory of Helmholtz… is consistent with observation’. Gold and Pumphrey did psychophysical experiments in which hearing thresholds were determined for listeners first for continuous pure tones and then for increasingly briefer stimuli of the same frequency. Gold and Pumphrey showed that their results could only be accounted for by considering the cochlea as a set of resonators, each of which responds to a narrow frequency range. In a second neat experiment, listeners had to detect differences between the sound of repetitive tone pips (series one) and those same stimuli but with the phase of every second pip inverted (series two, in which compressions replaced rarefactions and vice versa). Out-of-phase pips should counteract the action of in-phase pips and, following the child-on-swing analogy, rapidly bring swinging to a halt. Therefore, the argument goes, the two series should sound different. By increasing the silent interval between pips until the difference disappeared, the experimenters could infer how long the vibrations (or swinging) appeared to persist and could then put a measure on the quality factor (Q), or narrowness in frequency range, of the presumed underlying resonance. From the first experiment, Gold and Pumphrey derived values of Q of 32 to 300, meaning that the range of response was as little as 1/300-th of the imposed frequency—based on the picture of a broad travelling wave. The second experiment gave comparable results. However, their resonance interpretation has been dismissed because of a methodological flaw in the second experiment: the spectral signatures of the two series are not the same and provide additional cues. Nevertheless, it is not widely appreciated that the first experiment seems methodologically sound, and its results remain persuasive. I think the resonance theory deserves reconsideration. The evidence of my ears tells me that the cochlea is very highly tuned, and an active resonance theory of some sort seems to provide the most satisfying explanation. Furthermore, as well as Gold's neglected experiment, we now know from studies of acoustic emissions that the relative bandwidth of spontaneously emitted sound from the cochlea can be 1/1000 of the emission's frequency, or less. My research, guided by Professors M. V. Srinivasan and N. H. Fletcher, has centred on finding an answer to that most fundamental question: if the cochlea is resonating, what are the resonant elements? A point of inspiration for me is Gold's later discussion of cochlear function ( Gold 1987 )—some nine years after Kemp's discoveries had been made. Gold draws a striking analogy for the problem confronting the cochlea, whose resonant elements—whatever they are—sit immersed in fluid (the aqueous lymph that fills the organ). To make these elements resonate is difficult, says Gold, because they are damped by surrounding fluid, just like the strings of a piano submerged in water would be. He concludes that, to make ‘an underwater piano’ work, we would have to add sensors and actuators to every string so that once a string is sounded the damping is counteracted by positive feedback. ‘If we now supplied each string with a correctly designed feedback circuit,’ he surmises, ‘then the underwater piano would work again.’ My research is investigating what Gold's underwater piano strings might be. A suggestion put forward in a recent paper ( Bell and Fletcher 2004 ) is that resonance might occur in the space between the cochlea's geometrically arranged rows of outer hair cells. These cells are both effectors (they change length when stimulated) and sensors (their stereocilia detect minute displacements), so a positive feedback network can form that sets up resonance between one row of cells and its neighbour. The key is to transmit the feedback with the correct phase delay, and the new paper describes how this can be done using ‘squirting waves’ in the gap occupied by the outer hair cell stereocilia. The paper suggests that the outer hair cells create a standing wave resonance, from which energy is delivered to inner hair cells (where neural transduction takes place). In this way, the input signal is amplified before it is detected—an active system functioning just like Gold's regenerative receiver. With a prime candidate in place for the resonating elements, this should, I think, prompt us to re-evaluate resonance theories of hearing, which were first put forward by the ancient Greeks and which, irrepressibly, keep resurfacing. The best-known resonance theory was that formulated by Helmholtz, but at that time no satisfactory resonating elements could be identified, and it lapsed until Gold's attempt to revive it. There are other difficulties in reviving a resonance theory of hearing, but I think they can be overcome. If there really are resonant elements in the ear, the outstanding question would be, how are they stimulated? It is conceivable that motion of the conventional travelling wave sets them off, in which case we have an interesting hybrid of travelling wave and resonance. The other possibility, which I favour, is that outer hair cells are stimulated by the fast pressure wave that sweeps through all of the cochlear fluid at the speed of sound in water (1,500 m/s). If that is the case, and outer hair cells are primarily pressure sensors, not displacement detectors, then the ear is a fully resonant, pressure-driven system. New life, perhaps, to that old resonance idea.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521729.xml
551594	The role of the vomeronasal system in food preferences of the gray short-tailed opossum, Monodelphis domestica	Although feeding deficits have been reported in snakes and lizards following vomeronasal system disruption, no deficit has been previously reported in a mammal. We tested gray short-tailed opossums with items from four different food categories prior to occluding access to the vomeronasal organ. Preoperatively, opossums preferred meat to fruit or vegetables. Following occlusion of the nasopalatine canal, but not after control treatment, opossums failed to demonstrate food preferences.	The vomeronasal system (VNS) is a nasal chemosensory system usually considered primarily a pheromone-detecting system [[ 1 - 4 ] for recent reviews]. The system, present in most terrestrial vertebrates, is comprised of the peripherally situated vomeronasal organ (VNO), whose sensory neurons project their axons to the accessory olfactory bulb (Figure 1 ), and the further projections from the accessory bulb into the limbic forebrain [ 1 - 4 ]. Figure 1 Head of an opossum as viewed from the side illustrating the position of the vomeronasal organ, olfactory epithelium and their respective connections to the accessory olfactory bulb and main olfactory bulb. In snakes and lizards, in addition to its role in pheromone detection, the VNS is important for feeding behavior [ 5 - 7 ]. To date, no study has reported a feeding deficit in a mammal deprived of a functional VNS. It is possible that the failure to report feeding deficits in rats, mice and hamsters deprived of a functional VNS is a result of the fact that these animals have been fed laboratory diets over many generations. It is also conceivable that with the emphasis on the pheromonal function of the VNS, the issue of the role of the VNS in feeding behavior was overlooked. Gray short-tailed opossums, Monodelphis domestica , are members of the Didelphidae or American opossums. The didelphids are the most ancient marsupial family and thought to be the origin of all other New World and Australian marsupials [ 8 , 9 ]. M. domestica was recently introduced into laboratories [ 10 ] and has, therefore, not been fed a laboratory diet over many generations. As these opossums respond to a variety of foods [ 11 - 13 ], they are good models to investigate the role of the VNS in food preferences in mammals. Six male and seven female gray short-tailed opossums, 12–16 months old, were used as test subjects in this study. The opossums were progeny of animals originally purchased from the Southwest Foundation for Biomedical Research (San Antonio, Texas, USA) and were offspring of different parents. The opossums were housed individually in plastic cages (42 × 21 × 20 cm) with wood shavings as bedding, and provided with cylindrical plastic containers for nesting. The animals had dry fox food (Milk Specialties Co., New Holstein, WI, USA) and water available ad libitum . To determine which foods to use in the formal experiment, a preliminary test was conducted in which 24 different foods, representing meats, fruits and vegetables were presented to the opossums. Each opossum was presented with one food per day, selected at random, and its consumption of the food noted. The animals were allowed three minutes to investigate and eat the food, which was placed on an 8 × 20 cm cardboard tray at the front of the home cage. Each animal received two trials with each food. Of the 24 foods tested 16 were selected for the formal experiment based on the animals' observed preferences. During testing the opossums were simultaneously presented with four foods one from each of the following food groups: fruits (apples, oranges, peaches, cantaloupes), meats (mealworms, chicken, pork, crickets), processed vegetables (Raisin Bran, Cheerios, whole wheat bread, bagel) and unprocessed vegetables (corn, peppers, carrots, broccoli). The animals were tested daily with one of 16 food combinations (4 foods × 4 categories). Four sterile Petri dishes, each 3.5 × 1.0 cm, were placed equidistant from each other and glued to an 8 × 20 cm cardboard tray. One piece (0.21 cm 3 ) of food from each food category, was placed in a dish. Before each test session, feeding troughs containing fox food were removed from each opossum's cage. At the beginning of a trial the food tray was placed in the front of the animal's home cage and the behavior of the animal videotaped for three minutes using a digital video-camera (Sony DCR-VX2000, 30 frames/sec) placed at a distance of 3 m from the test cage. Trays, dishes and unconsumed food were discarded after each trial. After testing, food troughs containing fox food were replaced in the animals' home cages. Fresh disposable gloves were used when handling dishes, trays and foods to prevent transfer of food odors. Videotapes of food trials were analyzed using a videocassette recorder (JVC SR-VS30U), BTV Pro 5.4.1 and "Videoanalyzer" software (designed by John L. Kubie, Downstate Medical Center). For control surgery and occlusion of the nasopalatine canal, opossums were lightly anesthetized with Ketaset (0.25 cc/100 g, i.m.) and atropine sulfate (.05 cc/100 g, i.m.) Access to the vomeronasal organ (VNO) was blocked with gel foam and Crazy Glue™ (Elmer's Products, Inc., Columbus, OH) applied to the roof of the mouth, covering the opening to the nasopalatine canal. Cresyl violet crystals were added to the Crazy Glue to facilitate visualization of the block. Opossums were visually checked daily to insure that the VNO block remained in place. Control surgery consisted of placing Crazy Glue on the roof of the mouth to either side of the nasopalatine canal. Opossums were allowed two days to recover before postoperative testing. The VNO block remained in place for only one test day in all thirteen opossums. Therefore, pre-post operative and control comparisons were only made for trials identical in composition (foods tested) and order (position on the tray) to the first postoperative test day. Control postoperative trials were run as described above. All 13 animals were tested under both control and experimental (nasopalatine canal blocked) conditions, with the order of surgical treatment randomized. The first food selected and consumed on each trial was identified as the animal's food choice. Statistical analyses utilized a chi-square test of the frequencies of choices of each food type for pre-operative, control and experimental trials. Figure 2 depicts the results in terms of the percentage of choices of each food type under the three conditions. Figure 2 Percentage of food choice responses (first food type selected and consumed) by opossums prior to surgery (Preop), after control occlusion (Control) and after occlusion of the nasopalatine canal (Postop). Preoperatively, opossums preferred meats to fruits and fruits to processed and unprocessed vegetables (Figure 2 ). The most preferred food within the meat category was crickets, within the fruit category was cantaloupe, within the unprocessed vegetable category was corn and within the processed vegetable category was whole wheat bread. Whereas the preoperative comparison trials on the thirteen opossums revealed significant preferences among the food categories (χ 2 = 10.08, df = 3, p < .02), postoperatively, no significant difference in food category preference was observed (χ 2 = 0.85, df = 3, p > .05). During control trials the opossums continued to demonstrate significant food preferences (χ 2 = 9.46, df = 3, p < .05,). Meat was again, the most preferred food category (Figure 2 ). One deficiency of this study is that, because the opossums removed the glue blocks after the first day, we were unable to verify that the nasopalatine occlusion had, indeed, prevented access to the vomeronasal organ. However, we had previously [ 14 ] demonstrated, using the identical technique, that this method prevented access of substances to the VNO. Furthermore, the failure of opossums with nasopalatine canal occlusion to demonstrate the food preferences observed during preoperative and under control conditions, strongly suggests that the block was effective. This study suggests that without a functional VNS, the food preferences of gray short-tailed opossums are significantly impaired. Previous studies on the VNS of mammals have not addressed the issue of changes in feeding behavior. It remains to be seen whether other mammals, newly introduced into the laboratory, or bred over many generations in the laboratory, also demonstrate a food preference deficit when deprived of a functional VNS. Studies comparing wild populations with laboratory-reared populations might contribute information resolving this issue. It is not surprising that food preferences might be influenced by vomeronasal stimulation since the vomeronasal organ of many vertebrates, including opossums, is directly accessible from the oral cavity [ 2 ] via the nasopalatine duct. Thus, food in the mouth could be sensed by the vomeronasal system. Whether this mechanism is, in fact, utilized in food selection is not known and would have to be the subject of future investigation. It is likely, however, that the expression of food preference is a result of the interaction of multiple sensory systems including taste, olfaction, vision, trigeminal and vomeronasal. Authors' contributions MH conceived of and designed the experiment and wrote the manuscript YD conducted the experiment, extracted the data from the videotapes and analyzed the data statistically IZ supervised YD in the conduct of the experiment and instructed YD in the experimental procedures and data analysis	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC551594.xml
521073	Nosocomial outbreak of neonatal Salmonella enterica serotype Enteritidis meningitis in a rural hospital in northern Tanzania	Background Clinicians at Haydom Lutheran Hospital, a rural hospital in northern Tanzania noted an unusually high case-fatality rate of pediatric meningitis and suspected an outbreak of an unknown agent or an organism resistant to the empirical therapy. Methods We established a provisional microbiology laboratory to investigate the suspected outbreak. Blood and spinal fluid specimens were taken from children below the age of seven years with suspected meningitis. The blood and spinal fluid specimens were inoculated in commercial blood culture bottles and locally prepared Thayer-Martin medium in slanted tubes, respectively. The bacterial isolates were sent to Norway for further investigation, including susceptibility testing and pulsed-field gel-electrophoresis (PFGE). Results Among 24 children with suspected meningitis and/or septicemia, five neonates had meningitis caused by Salmonella enterica serotype Enteritidis, all of whom died. Two children had S . Enteritidis septicemia without meningitis and both survived. Genotyping with PFGE suggested a clonal outbreak. The salmonella strain was resistant to ampicillin and sensitive to gentamicin, the two drugs commonly used to treat neonatal meningitis at the hospital. Conclusion The investigation reminds us that nontyphoidal salmonellae can cause meningitis associated with very high case-fatality rates. Resistance to multiple antimicrobial agents increases the risk of treatment failure and may have contributed to the fatal outcome in all of the five patients with salmonella meningitis. The investigation indicated that the outbreak was nosocomial and the outbreak subsided after hygienic measures were instituted. Establishing a provisional microbiological laboratory is a valuable and affordable tool to investigate and control outbreaks even in remote rural areas.	Background Nontyphoidal salmonellae are a common cause of food-borne illnesses. In Africa, nontyphoidal salmonellae are the most common cause of bloodstream infections in children younger than five years [ 1 ]. While meningitis caused by nontyphoidal salmonellae is uncommon in economically developed countries [ 2 ], it is more frequent in tropical countries, particularly in children younger than six months, and associated with higher case-fatality rates than meningitis caused by other bacteria [ 1 , 3 - 5 ]. At Haydom Lutheran Hospital, a rural hospital in northern Tanzania, clinicians noted an extraordinarily high case-fatality rate (>60%) from pediatric meningitis in the period January 1998 to April 2000. It was suspected that the high case-fatality rate of meningitis was due to an outbreak of an unusual etiological agent, or an organism resistant to the hospital's standard empirical treatment, which was ampicillin and gentamicin for infants (<2 months) and penicillin and chloramphenicol for older children. Thus, we established a provisional microbiology laboratory to identify the causative agents and to facilitate the implementation of effective preventive measures. Methods From a total of 360 children admitted from July to August 2000, blood and/or spinal fluid specimens were collected from 24 children aged one day to six years (median age 23.5 days) with suspected meningitis and/or septicaemia, after careful evaluation by the attending pediatrician. Blood and spinal fluid specimens were inoculated in BBL SeptiChek blood-culture bottles (Becton Dickinson, Sparks, MD USA) and on locally prepared non-selective Thayer-Martin medium in slanted tubes, respectively. All cultures were incubated at 35°C for 5 days and inspected daily for bacterial growth. Positive bacterial specimens were shipped to Institute of Medicine, Haukeland University Hospital, Norway, for further study. The total cost of the laboratory reagents used on site was $475. Positive specimens were identified using standard laboratory methods [ 6 ]. The susceptibilities of the isolates to antimicrobial agents were examined by disk diffusion method on PDM medium (AB Biodisk, Solna, Sweden) [ 7 ]. The isolates were genotyped with pulsed-field gel electrophoresis (PFGE). Statistics were calculated with Stata 8 for Mac OSX (Stata Corporation, College Station, TX). Results On clinical grounds, twenty-four children were considered to have possible sepsis or meningitis. Blood culture was taken from 23 children, but 13 had received prior antibiotic treatment. Spinal fluid culture was taken from 16 children, of whom ten had received prior antibiotic treatment. Both blood culture and spinal fluid culture was obtained from a total of 15 children. Salmonella enterica serotype Enteritidis was isolated from seven patients, of whom four had positive cultures from both blood and spinal fluid and three from blood only. One of the three patients, who had S . Enteritidis isolated only from blood culture, had pus in the spinal fluid, and was therefore considered a case of S . Enteritidis meningitis, resulting in a total of five cases of S . Enteritidis meningitis. One isolate each of Staphylococcus aureus and Streptococcus pyogenes were isolated from spinal fluid and three isolates of coagulase-negative staphylococci were isolated from blood. Antimicrobial susceptibility testing showed that all the S . Enteritidis isolates were resistant to chloramphenicol and ampicillin, intermediate resistant to cefuroxime, and sensitive to gentamicin, cefotaxime and ciprofloxacin. As shown in Figure 1 , the PFGE patterns were identical for all the eleven S . Enteritidis isolates except one, which differed by only one band (Figure 1 shows a total of 17 strains, 6 of which are duplicate isolates from the same patients). All children with S . Enteritidis infections were neonates (median age 15 days, range: 8 to 27 days), whereas those with other infections or negative cultures, on average, were older (median age 4 months, range: 1 day to 6 years). All children with S . Enteritidis infections had been delivered at Haydom Lutheran Hospital, and five out of the seven never left the hospital before they became ill. Table 1 shows the characteristics of the patients with S . Enteritidis infection. All five children with S . Enteritidis meningitis died. The two children with S . Enteritidis sepsis survived. Five (29%) of the 17 children without verified S . Enteritidis infection also died. Among the 24 children investigated, S . Enteritidis meningitis was associated with a relative risk of 3.8 (95% confidence interval 1.8 to 8.1) for fatal outcome. Discussion The finding of an outbreak of bacteremia and meningitis caused by S . Enteritidis was not anticipated. Genotyping with PFGE suggested a clonal outbreak. This genotyping information, the susceptibility patterns and the clinical information that all children with S . Enteritidis infections were born at the hospital and that the majority never left the hospital before they became ill, strongly suggest that the outbreak was nosocomial. Nontyphoidal salmonella infections are frequently associated with animal reservoirs and infection usually originates from food products. Nosocomial spread of nontyphoidal salmonellae, particularly in neonatal wards, is known from the literature [ 8 ]. Neonates are at particular risk of infection because of relatively reduced gastric acidity and peristalsis [ 1 ]. In previous nosocomial outbreaks caused by salmonellae, the sources of infection have been related to the use of contaminated medications, diagnostics, blood products, banked human milk, the use of raw eggs or yeast in tube feeding and improper disinfection of devices such as rubber tubes for oropharyngeal suction [ 8 , 9 ]. The exact source of the outbreak at the hospital was not established. Direct food-borne transmission was unlikely, since the neonates at the hospital were fed on breast milk. Possible sources include contaminated instruments, clothes or bathing facilities for the newborn. Spread of infection may have occurred by baby-to-baby transmission or via family members and/or hospital staff. The medical staff at the hospital was informed about the findings, and immediate interventions in the form of hygienic measures were instituted, including the reinforcement of disinfection and hand-washing practices. Data from the hospital annual reports shows that the case-fatality rate from pediatric meningitis dropped from >60% before the intervention to 40% by 2001. The S . Enteritidis strain responsible for the outbreak was resistant to two of the first-line drugs, ampicillin and chloramphenicol, but sensitive to gentamicin. This finding is not surprising, since multi-drug-resistant S . Enteritidis has been reported from this region for decades [ 10 ]. The children at the hospital were treated with a combination of ampicillin and gentamicin. The high case-fatality rate in these patients implies that de facto monotherapy with gentamicin may be suboptimal as treatment for S . Enteritidis meningitis considering that gentamicin traverses the blood-brain-barrier poorly, is bound to proteins and inhibited by the acidity in infected cerebrospinal fluid. However, it also reflects the poor prognosis of neonatal S . Enteritidis meningitis regardless of therapy. Molyneux reported fatal outcome for 58% of children with meningitis caused by nontyphoidal salmonellae despite routine treatment with chloramphenicol, to which all bacterial isolates were sensitive in vitro [ 4 ]. Many authorities recommend third-generation cephalosporins as empirical chemotherapy for meningitis caused by gram-negative bacteria [ 11 ], not only because of high bactericidal activity due to low minimum inhibitory concentrations (MICs), but also because they penetrate the blood-brain-barrier better than both gentamicin and chloramphenicol. A third-generation cephalosporin, such as cefotaxime would have been an excellent therapeutic option in this case. However, the price of the newer cephalosporins is often prohibitive in the setting of low-income countries such as Tanzania. Ciprofloxacin is generally not recommended for use in children due to potential adverse effects, but can be resorted to for treatment of life-threatening infections with multidrug-resistant nontyphoidal salmonellae [ 12 ]. The standard empirical treatment regimen at the hospital could not be changed due to financial constraints. Infections caused by nontyphoidal salmonellae in children in Africa are more common during the rainy season and have been associated with malaria, anemia and malnutrition [ 1 ]. However, there is considerable overlap between these medical conditions, all of which may be more frequent during the rainy season. This investigation was performed during the dry season. Infections caused by nontyphoidal salmonellae have also been associated with HIV infection [ 1 ]. We do not know the HIV status of the children involved in this outbreak, however, in this part of Tanzania, the rate of HIV infection is less than 0.5% [ 13 ]. Conclusions There are a number of lessons to be learned from this investigation. We are reminded that S . Enteritidis can cause meningitis, which carries a very high case-fatality rate [ 1 , 3 , 4 ]. Our findings support former reports that S . Enteritidis can easily spread by nosocomial transmission, particularly in neonatal wards. Resistance to multiple antimicrobial agents increases the risk that empirical therapy will fail, especially in settings where modern cephalosporins are not affordable. Without adequate laboratory facilities, correct diagnosis and treatment of bacterial meningitis and septicemia in children remains a challenge. However, the report also shows that establishing a provisional microbiology laboratory can be a valuable and affordable tool to investigate and curb epidemics even in the setting of remote rural Africa, provided there is a proficient laboratory willing to assist. Competing interests None declared. Authors' contributions HV was the principal investigator, participated in the planning and execution of the work, including performing the on-site microbiological procedures, and analysis of data, and was the main responsible author. BB participated in planning, microbiological investigations in Norway, data analysis and writing. CK and NN undertook clinical investigation and sample collection and participated in the writing. RJ was responsible for the microbiological investigations in Norway and participated in the data analysis and writing. NL was the project coordinator and participated in planning, data analysis and writing. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521073.xml
548953	Discovery-Based Science Education: Functional Genomic Dissection in Drosophila by Undergraduate Researchers	How can you combine professional-quality research with discovery-based undergraduate education? The UCLA Undergraduate Consortium for Functional Genomics provides the answer	The excitement of scientific research and discovery cannot be fully conveyed by didactic lectures alone. Several recent initiatives and proposals, therefore, have supported a more participatory, discovery-based instruction for undergraduate science education [ 1 , 2 ]. In functional genomics, we have found an ideal platform to simultaneously benefit students and contribute to scientific discovery. The sequencing of eukaryotic genomes has facilitated the identification of complete sets of genes in humans and model genetic organisms. This has allowed many forms of high-throughput analyses of transcriptional profiles, protein interactions, structural motifs, and even genome-wide knock-downs in cell lines or in selected organisms. However, one of the best tools to provide functional information about gene action— obtaining in vivo evidence about the phenotype resulting from heritable loss of function—is difficult and less amenable to high-throughput research. We were able to achieve a large-scale in vivo analysis with a significant number of undergraduate students at UCLA, called the UCLA Undergraduate Consortium for Functional Genomics. This work, a practical manifestation of policy positions proposing discoverybased education, is described in summary form here (and in Box 1 ) and in detail online at http://www.bruinfly.ucla.edu . This effort combines professional-quality research with a strategy for research-based undergraduate education. Box 1. Scientific Results The Drosophila eye is an intricate neurocrystalline lattice of approximately 800 individual ommatidia arrayed in a very precise order ( Figure 3A ) [ 3 ]. Minor perturbations in ommatidial development can be easily detected, making it a very sensitive system for functional genomic screens [ 3 ]. Our study utilized 1,375 unique recessive lethal transposable element (P-element) insertion stocks from the 2nd and 3rd chromosomes of Drosophila to characterize their later role in eye development. To avoid early lethality, the FLP/FRT system was used to generate homozygous mutant tissue specifically in the eye [ 4 ]. Of the mutations analyzed, 501 (36%) displayed a mutant eye phenotype, providing the first genome-wide estimate of the fraction of essential genes that are also involved in eye development. Adult eye phenotypes were classified into three broad classes: rough, cell lethal, and glossy ( Figure 3 ). The genes responsible for these phenotypes were assigned into 19 different functional categories, which are summarized in Table 1 . Signal transduction components previously established to be important for eye development (e.g,. EGFR , pointed , Star , tramtrak , Delta ) were identified, validating the effectiveness of our screen. In addition, our genomics approach has shown that a number of novel classes of genes are involved in eye development that have not been previously described ( Table 1 ). Figure 3 Summary of Phenotypes Determined Light (left panels) and scanning electron (right panels) micrographs of mosaic Drosophila eyes. Large homozygous mutant clones are orange (arrowheads); heterozygous tissues are dark red. Examples are shown of lethal mutations that give a (A) wild-type (63.4% of lethal mutations), (B) rough (disordered ommatidia, 18.2%), (C) cell lethal (absence of homozygous mutant tissue, 14.5%), and (D) glossy (loss of lens structure, 3.9%) phenotype. For details on how clones are generated, see http://www.bruinfly.ucla.edu . Table 1 Summary of Genes Involved in Adult Eye Development Molecular information is available for 315 of the 501 phenotypic mutants. Of these, 291 were identified as unique genes and categorized into 19 classes, based solely on the insertion site data from FlyBase We have created a novel curriculum with three main components: didactic, computer, and laboratory. The only prerequisite for this course is high school advanced-placement-level biology; all other knowledge necessary for the course is taught within it. Since there are no other prerequisites for the course, a majority of the students enrolled are freshmen and sophomores, enabling us to educate them in this novel way early in their undergraduate career. Approximately 30 students take this course each quarter, and it is offered every quarter through the school year, allowing for a broad impact. In the lecture series, students are exposed to interactive lectures on background material, basic concepts of genetics, research ethics, and career options. For their “midterm,” each student proposes an experiment in a grant proposal formatted according the National Institutes of Health requirements. The “final” is written as a scientific paper summarizing the student's own results. In the computer section, students perform research with a “virtual fly lab” to help them understand more about their crosses in the laboratory section. In addition, they learn about modern genomic resources available on the Internet, and utilize some of the genomics tools available (e.g., BLAST) to help them determine the identity and function of their disrupted genes. The main component of the class, however, is the laboratory portion. In the laboratory, the students perform all the necessary work to manipulate the genotypes of their stocks to determine what effect homozygous mutation of their target genes has in the adult Drosophila eye ( Figure 1 ). To accomplish this, the students perform five-generation Drosophila crosses that nicely fit into a ten-week quarter. Each student is assigned about ten mutants to work with. During the quarter, students are able to recombine each mutation onto a flippase (FLP) recombination target (FRT) chromosome, generate mutant somatic clones, and record details of the adult eye phenotype with both light and scanning electron microscopic techniques. The students then upload their data into an online database ( http://www.bruinfly.ucla.edu ). Figure 1 Representative Pictures from the Laboratory Section of the Course Our database contains pictures of the mutant eyes for all of the stocks examined, as well as other information pertinent to that stock, including the gene disrupted, the exact genomic location of the P-element insertion, and whether an excision of the P-element has been performed and its results. A sample Web page of the database is shown in Figure 2 . Following the introductory course, a small number of students continue to analyze the developmental basis for select mutations in future quarters in more advanced laboratory classes. In these advanced classes, the students perform P-element excision experiments to determine whether the mutant phenotype observed is indeed derived from the P-element. These students have performed 294 excision experiments, the results of which indicate that 72% of the stocks successfully revert to wildtype phenotype when the P-element is removed. Over the last two years, we have educated 138 students in the introductory course. Advanced classes have totaled 96 student-quarters (46 students, each working two or more additional quarters). Figure 2 Example of the Type of Data Available from the Online Database ( http://www.bruinfly.ucla.edu ) In summary, discovery-based experiments in functional genomics are well suited for undergraduate education: they actively engage a large number of students in research without compromising their didactic training. The sense of ownership developed from this research amplifies the students' learning experience. For the research community, the online database and the large collection of newly generated FRT-lethal lines represent a valuable resource for future experiments in eye development. Furthermore, the stocks developed can be used to create mutant clones in an investigator's tissue of choice. This novel approach for performing research, for which functional genomics is very amenable, not only encourages many students in new ways of thinking but also generates professional-quality results and resources for the scientific community. Supporting Information Figure S1 A Compilation of the Undergraduates and Some of Their Fly Mutants (1.2 MB JPG). Click here for additional data file.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548953.xml
528725	Caryoscope: An Open Source Java application for viewing microarray data in a genomic context	Background Microarray-based comparative genome hybridization experiments generate data that can be mapped onto the genome. These data are interpreted more easily when represented graphically in a genomic context. Results We have developed Caryoscope, which is an open source Java application for visualizing microarray data from array comparative genome hybridization experiments in a genomic context. Caryoscope can read General Feature Format files (GFF files), as well as comma- and tab-delimited files, that define the genomic positions of the microarray reporters for which data are obtained. The microarray data can be browsed using an interactive, zoomable interface, which helps users identify regions of chromosomal deletion or amplification. The graphical representation of the data can be exported in a number of graphic formats, including publication-quality formats such as PostScript. Conclusion Caryoscope is a useful tool that can aid in the visualization, exploration and interpretation of microarray data in a genomic context.	Background The application of high-throughput technologies (such as DNA microarrays) to biomedical experimentation generates large quantities of data that can be difficult to browse and interpret in the absence of a graphical representation. Eisen et al. have previously displayed clustered microarray data using a false color representation that greatly aids in the intuitive interpretation of the data ([ 1 ]). However, when these data are from array comparative genome hybridization (arrayCGH) experiments ( e.g. , see [ 2 ]), the genomic locations of the reporters (the molecules in each spot on a microarray) that were used to generate the data are important for interpretation. A relative increase or decrease in the ratios for a group of reporters that report on adjacent genomic locations may indicate amplification or deletion of that genomic region, respectively. Additionally, even the analysis of expression data in the context of genomic position can also identify regions of amplification or deletion, or even cases of aneuploidy ([ 3 , 4 ]). In addition to being able to view and browse arrayCGH data, it is also important that the data be readily connected to annotation sources, such that a user can easily determine the identity and attributes of the gene represented by a reporter that was present on a microarray, which for instance may show evidence of amplification or deletion. For example, in arrayCGH experiments using tumor cells as the DNA source, there is an obvious value in rapidly determining whether a deleted region contains a tumor suppressor gene. Finally, researchers frequently need to create figures, for publications, communication with co-workers, supplemental websites, or presentations. Thus researchers should be able to produce the visual representation of their data in a variety of graphic formats. Caryoscope was originally implemented as a Web form, generating either a bitmap or a clickable PDF output. When this became an important day-to-day tool for our users ([ 5 - 9 ]), we created an improved, interactive version, consisting of a standalone application for analyzing arrayCGH data and an open architecture of re-usable classes that may be embedded by other developers in their own applications. In this paper, we focus on Caryoscope as an application. Some other software packages were developed while this work was in progress, and can perform some of these functions. For instance, Genome2D ([ 10 ]) is designed to display bacterial transcriptome data on linear chromosome maps, while SeeCGH ([ 11 ]) was designed for viewing arrayCGH data (only for 2-channel arrays). However, both of these programs are designed to run solely on the Windows operating system, whereas Caryoscope is a Java application that can be used on Macintosh OS X, Linux and various UNIX operating systems, as well as Windows. Greshock et al. ([ 12 ]) have built similar functionality, called CGHAnalyzer, on TIGR's Multiple Experiment Viewer (MeV) platform, but with a different (circular) whole-genome view. Furthermore, Caryoscope can be run in a command line mode, making it easy to embed within a CGI or a processing pipeline. Implementation We implemented Caryoscope in Java ([ 13 ]) and deployed it as a Java Web Start ([ 14 ]) application, so a user may run it directly from our website ([ 15 ]) by clicking on a link. One can also install Caryoscope directly on a computer, but we recommend launching via the website in order to obtain the most current version of the software. Caryoscope accepts data as text input files in simple formats so as to maximize interoperability with other systems. Results Application features As input, Caryoscope accepts a single file in either the General Feature Format (GFF, [ 16 ]) or a tab-delimited (TXT) or comma-delimited (CSV) spreadsheet-compatible format. This file describes the chromosomes to be displayed, and a set of loci on the chromosomes annotated by a number of associated microarray datasets and other descriptive information. The structure of a Caryoscope input file is illustrated in Figure 1 . Once the user opens a file, Caryoscope automatically displays one of the datasets contained therein (Figure 2 ). Caryoscope displays each feature as a rectangle on the chromosome axis; the size of the rectangle on the horizontal axis, perpendicular to the chromosome, represents the magnitude of the associated data value, while the size of the rectangle in the vertical direction, along the chromosome axis, represents the size of the represented feature, based on its genomic coordinates. Pursuant to convention, the default display of Caryoscope represents positive values in red bars, which are drawn to the right of the chromosome, and negative values in green bars (though these colors can be changed), which are drawn to the left of the chromosome. Thus, based on color, size, and location, researchers can easily intuit the meaning of the graphical representation of their data. Caryoscope provides several modes in which the user may view the data; these are controlled by the View modes toolbar (Figure 2 ). In the various panning and zooming modes, the user may change the view of the data to drill down to specific regions of interest. In Navigate mode, the user sees tooltips (small informational pop-up windows) that appear immediately when mousing over the features, and can navigate to related URLs by clicking on each feature. Typically, users at Stanford University (our primary source of testers and users) link GenBank accessions, associated with the cDNA clones that are on their microarrays, to SOURCE Gene Pages ([ 17 ]). The zooming paradigm in Caryoscope is somewhat novel in that it permits independent control of the zoom scales in the X and Y directions (Figure 3 ). It allows users to select the best scaling to see detail along the chromosome axis, and the data values perpendicular to the chromosome axis, for their specific data. The Reset viewpoint button on the View modes toolbar (Figure 2 ) allows the user to return quickly to the default scaling. The behavior of Caryoscope in Navigate mode is shown in Figure 4 . The tooltip and URL text are computed for each feature by substituting the value of its annotations into the Feature tooltip expression and Feature URL expression settings, as illustrated in the figure. These features allow users to have immediate access to information about each feature as they browse the data. The user can enable two built-in computations on the data values: a user can compute the logarithm of the values (to any base specified by the user), and a user can compute a moving average of the values. Both these computations can be controlled from the Settings dialog (see Figure 2b ). Users can perform other computations outside Caryoscope; this is facilitated by the fact that we support common spreadsheet-compatible file formats (TXT and CSV). To prepare diagrams, the user can export the Caryoscope display to a variety of graphics formats via the Export dialog. Specifically, Caryoscope supports vector ( e.g. , PostScript and PDF) output for scalable publication-quality results, and raster ( e.g. , JPEG and PNG) output for ease of viewing, posting on supplemental websites, and inclusion in presentations. A user may export graphics from Caryoscope via the command line mode, without having to invoke the interactive user interface. For example, to export a view of a dataset as a PDF file, the user could invoke Caryoscope as follows: java -jar caryoscope-run.jar -inputFile 3395-2004-04-04.csv -visibleDataset RAT2_MEAN -outputFile 3395.pdf -outputFormat pdf All settings may be modified via the command line. The -listFormats option provides a list of available graphics output formats, and the -help option prints a brief summary of the options. A comparison of the features of Caryoscope with other, similar software is presented in Table 1 . Obtaining Caryoscope In addition to immediately executable copies, the complete source code for Caryoscope is available without limitations from our website ([ 15 ]), and is covered by a very liberal Open Source ([ 18 ]) license (the MIT License, [ 19 ]). All external components used by Caryoscope are also Open Source. We update Caryoscope frequently (approximately once every three weeks) and post news items on the website. We also send e-mail announcements to people who have requested them. Discussion Biological context Caryoscope is useful for viewing both arrayCGH and expression data in the context of genomic position. It helps a biologist gain insight by providing a high-level view of a large amount of data at once, where patterns can be perceived at a glance. A biologist studying amplifications or deletions in tumor cells may create and export graphics representing arrayCGH and expression data for the same cells using Caryoscope, and visually compare the two side-by-side. For instance, co-located regions that are amplified at the DNA level and over-expressed at the RNA level would provide excellent confirmation of the results. Using the zooming and panning features of Caryoscope, the biologist could focus on specific regions of interest. To identify regions of aneuploidy, the biologist can again simply examine the data visually. In this case, however, one would look for a large-scale pattern. One might specify a Minimum feature width of, say, 2 pixels (Figure 5c ), to ensure that any deletion or amplification, no matter how small in genomic coordinates, is easily visible. Rather than zooming in on specific regions, a researcher would tend to compare overall views of the entire genome. If it seems like practically all of one chromosome is amplified or deleted, the biologist would have strong evidence for aneuploidy. In a gene expression study, a biologist may suspect that some expression patterns are correlated with genomic position. Caryoscope allows one to view expression data, either for the whole genome or on a region of interest, to help confirm or refute a hypothesis. Finally, in all this work, the biologist may want to have quick and easy access to information about the genomic features displayed. As long as the information needed is available from the annotations that were saved in the input data file (or available at a URL that can be built based on the annotations), one can use the Feature URL expression and Feature tooltip expression (Figure 4 ) settings to provide immediate mouse-over feedback with this information – almost as if the application were customized for a specific field of interest. Software context We intend Caryoscope to be a bench-top visualization tool that biologists can use immediately to get day-to-day work done, with a very low "cost of entry" for getting started. This led us to a number of design choices. Caryoscope can be launched from our web site without a prior installation step. Since it is Open Source, anyone, including any for-profit organization, can use it without restrictions and without having to obtain a license or register for access. The input file formats and output graphical formats we chose are all in common usage. In particular, the TXT and CSV input formats can be generated using any popular spreadsheet or database software, or even with a plain text editor, without having to do any programming. Finally, we built Caryoscope to be content-neutral, with no hard-coded specificity to any research field. Thus, users of Caryoscope may control how annotations are treated as numerical data, and can "program" data-driven interactive behavior of the display ( i.e. , the tooltips and hyperlinks). Future work From the outset of this project, our biggest challenge has been how to accurately represent the huge amounts of data in a typical gene expression or arrayCGH experiment using the limited number of pixels available on the screen or on a printed page. If we skew our display algorithms too much towards producing a "sharp", high-contrast plot, we risk obscuring detail in the data and leading biologists to the wrong conclusions. On the other hand, since the size of the data elements, properly scaled from genomic coordinates to the display device, can be far less than the size of one pixel, we need a supportable way to "summarize" the data within each pixel and represent that summary as a single value: the color (including the brightness) of that pixel. Modern computer graphics systems use a technique called "anti-aliasing" ([ 20 ] and Figure 5a ) to render sub-pixel details with the illusion of smoothness. The Java subsystems we use in Caryoscope do this automatically and, in the current version of the software, we simply rely on them (Figure 5b ). However, the anti-aliasing in Java is designed to display visually appealing text, lines and arcs, but not to ensure the most accurate possible on-screen rendering of scientific data. Specifically, at low magnification, the data almost disappear unless we force a minimum pixel size for each locus (Figure 5c ). We will develop our display methods further to ensure that we can provide an easy-to-read display while retaining the subtle variations in the data. Following the spirit of medical diagnostic imaging (DICOM, [ 21 ]), whereby incorrect details in a few pixels could lead to an incorrect conclusion, we must ensure that our displays, which are used for important research decisions, are never misleading. One solution, suggested by [ 12 ], is to display the data elements, not aligned to the position of the loci along the chromosome, but rather in strict sequential order with a fixed width. While this solves the anti-aliasing problems, it does eliminate consistent chromosomal positions and alignments of the data. Furthermore, it causes the appearance of the display to be dependent on the specific choice of clones – which can be another source of subtle variation when comparing multiple datasets. Another idea is to display dots, rather than horizontal bars, so that the "spread" of the data is more visible even if data points are super-imposed. We will investigate this for a future release of Caryoscope. We are particularly concerned about the use of Caryoscope (or similar) graphics in vector formats (such as PostScript, PDF and SVG) that are subsequently rendered on diverse display devices and printers. Since we have no control over the rendering at the destination, it is likely that the same vector output could look very different on different devices. Once we have studied this problem in more detail, we intend to provide practical usage guidelines for researchers. Our experience with the application, and how it is used, leads us to believe that the current zooming paradigm should be revisited. While the model of a continuously zoomable 2D space provides users with the features they need, it can lead to displays whereby the data in specific regions of adjacent chromosomes are juxtaposed, even though their being next to each other is not intrinsically meaningful (see Figure 6 ). (An exception to this might occur in telomere amplification, or perhaps special behavior around the centromeres. In this case, the ability to align the chromosomes at either end, or at the centromeres, would be helpful.) In the future, we will modify our display so that the user can turn "on" or "off" the display of the available chromosomes. Within that display, and with the help of our users, we will review the role of the X-axis scaling: perhaps it should change the scaling of the data, or perhaps it does not belong in Caryoscope-like applications at all. A common user request is to display two or more arrayCGH and/or expression datasets side by side, either to determine regions of recurrent deletion or amplification, or to discern visually the impact that changes in chromosome copy number may have on transcript levels ( e.g. , see [ 5 ]). Another frequent request is to show the cytoband information for each chromosome. We intend to add these features as part of our future development, in the course of which we would perhaps redefine, or extend, the manner in which our input data are defined ( i.e. , we would accept the definition of the chromosome names, lengths and cytobands in a separate file that would be re-used by different datasets). Summary Caryoscope currently provides a flexible method to visualize, explore and create images of microarray data in a genomic context. With such a tool, microarray researchers will be able to answer questions about how genome copy number or genome position plays a role in biological processes or human diseases. Conclusions Caryoscope is a useful, flexible Java application for the visualization of microarray data in a genomic context. It is available as Open Source under the permissive MIT License, allowing anyone to use or modify it. Availability and requirements Project name: Caryoscope Project home page: Operating system(s): Platform independent Programming language: Java Other requirements: Java 1.4 or higher License: MIT License Any restrictions to use by non-academics: None Authors' contributions IABA compiled the functional requirements for the Java version of Caryoscope and designed and implemented the software. CAR conceived and wrote the original Perl version of Caryoscope. THB aided and participated in the design of the study, dealt with user support and worked with users to get feedback and suggestions for further development. CAB aided and participated in the design and evaluation of the study and provided feedback and suggestions for future development. GS supervised and participated in the design of the study. All authors read and approved the final version of the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC528725.xml
514553	Mental health first aid training in a workplace setting: A randomized controlled trial [ISRCTN13249129]	Background The Mental Health First Aid training course was favorably evaluated in an uncontrolled trial in 2002 showing improvements in participants' mental health literacy, including knowledge, stigmatizing attitudes, confidence and help provided to others. This article reports the first randomized controlled trial of this course. Methods Data are reported on 301 participants randomized to either participate immediately in a course or to be wait-listed for 5 months before undertaking the training. The participants were employees in two large government departments in Canberra, Australia, where the courses were conducted during participants' work time. Data were analyzed according to an intention-to-treat approach. Results The trial found a number of benefits from this training course, including greater confidence in providing help to others, greater likelihood of advising people to seek professional help, improved concordance with health professionals about treatments, and decreased stigmatizing attitudes. An additional unexpected but exciting finding was an improvement in the mental health of the participants themselves. Conclusions The Mental Health First Aid training has shown itself to be not only an effective way to improve participants' mental health literacy but also to improve their own mental health. It is a course that has high applicability across the community.	Background In 2000 we developed a Mental Health First Aid course in response to the findings of two large national mental health surveys in Australia [ 1 , 2 ]. These findings included a high prevalence rate of mental health problems (approximately 20% of adults in any one year), the poor mental health literacy of members of the Australian public (poor recognition and knowledge of symptoms and causes of mental health problems, where to seek help and what are the most effective treatments) and the widespread stigma towards people with mental health problems. Regular first aid courses are recognised as improving the public's giving of initial and appropriate help at medical emergencies but, unfortunately, most of these courses do not include mental health problems. The Mental Health First Aid course consists of three weekly sessions of three hours each. The content covers helping people in mental health crises and/or in the early stages of mental health problems. The crisis situations covered included suicidal thoughts and behavior, acute stress reaction, panic attacks and acute psychotic behavior. The mental health problems discussed included depressive, anxiety and psychotic disorders. The co-morbidity with substance use disorders is also covered. Participants learn the symptoms of these disorders, possible risk factors, where and how to get help and evidence-based effective help. The initial evaluation trial of the Mental Health First Aid course was an uncontrolled one with 210 members of the public with pre, post and 6-month follow-up. This trial showed that participants improved: their recognition of mental disorders, their beliefs about what treatments were helpful, attitudes towards people with mental illness, the amount of help provided to people with mental health problems, and their confidence in providing help to these people [ 3 ]. The next step in our evaluation of this course was to conduct a randomised trial involving a wait-list control group. The present article reports this study, which was carried out in a workplace setting. Methods Participants Eligible participants (approximately 4800) were all Canberra-based employees of two Australian government departments: Health and Ageing, and Family and Community Services. The trial was advertised to staff by email. Participants had to agree to be randomly assigned to receive the training in either Month 1 or Month 6. Training was delivered and data collected at the worksite during office hours. Interventions The course content has been described in the Background and previously [ 3 ] and further details can be found at the Mental Health First Aid website [ 4 ]. The training followed set lesson plans and all participants were given a Mental Health First Aid Manual to keep [ 5 ]. Training was administered at the worksite in classes of 6–18 participants. Participants did not necessarily stay in the same class, but moved between classes to complete the course as necessitated by their work schedule. One instructor carried out all the training. She is the developer of the Mental Health First Aid course and had trained over 1000 people before the start of the trial. Participants received training either immediately (June) or after a five-month delay (November). Those who received training immediately constituted the intervention group and the wait-listed group was the control. To monitor whether the intervention was actually received, an attendance roll was kept for each class. Objectives The main objective was to assess whether Mental Health First Aid training improved mental health literacy and helping skills relative to a wait-list control. A secondary objective was to assess any benefits to the participants' own mental health. Outcomes Outcomes were measured in the month before intervention (the pre-test assessment) and in the fifth month after intervention (the follow-up assessment). The intervention group received training in Month 1 (immediately after pre-test) and the wait-list control group received training in Month 6 (immediately after the follow-up). All outcomes were measured by self-completed questionnaires based on the ones used in the uncontrolled trial of Mental Health First Aid [ 3 ]. The pre-test questionnaire (see Additional File 1 ) covered the following: socio-demographic characteristics of the participant, why they were interested in doing the course, history of mental health problems in participant or family, confidence in providing help, contact with people who have mental health problems in previous 6 months and help offered, recognition of a disorder in vignettes describing a person with depression and one with schizophrenia, belief about the helpfulness of various interventions for the persons described, a social distance scale to assess stigmatizing attitudes [ 7 ], and whether the participant or a family member or friend had ever had a problem like the one in the vignette. To score the items on beliefs about treatment, a scale was created showing the extent to which participants agreed with health professionals about which interventions would be useful. For depression, there is a professional consensus that GPs, psychiatrists, clinical psychologists, antidepressants, counseling and cognitive-behavior therapy are helpful [ 6 ]. Thus, participants received a score from 0 to 6 according to the number of these interventions endorsed as helpful and this was converted into a percentage. For schizophrenia, there is a professional consensus that GPs, psychiatrists, clinical psychologists, antipsychotics and admission to a ward are helpful for schizophrenia [ 6 ]. "Helpful" ratings were summed to give a score from 0 to 5 and converted to a percentage. The questionnaire ended with the SF-12, which provided scales assessing the participant's mental and physical health [ 8 ]. These scales were scored using Andrews' [ 9 ] integer scorer. The follow-up questionnaire was the same as the pre-test questionnaire except that it omitted the sociodemographic questions and asked about contact with anyone with a mental health problem over the 5 months since the last questionnaire (rather than 6 months). The questionnaires were sent out via internal departmental mail by a human resources staff member in each place of employment. The questionnaires were completed anonymously with only an ID number and posted back to the researchers at the Centre for Mental Health Research. The IDs of any non-responders were sent back to the human resources staff member who sent out a reminder. The researchers were never told the names of individual respondents and the human resources staff member in the place of employment never saw any completed questionnaires or individually identifiable data. Sample size The study was planned to have a sample of 300. The sample size was determined by practical constraints: when it was convenient to run classes that fitted the employees' work schedule and the workload on the instructor. It was determined that this sample size had excellent power to detect medium effect sizes for both continuous and dichotomous outcomes [ 10 ]. The trial was originally planned to involve only one workplace, but was extended to a second one because the number of participants recruited was smaller than expected. The lower recruitment appeared to be due to the requirement that participants agree to random assignment to training at either of two periods. Randomization and blinding A staff member in the human resources section of the place of employment kept a list of participants' names and ID numbers. The researchers only had access to the IDs. One of the researchers (Jorm) randomly assigned participants to training or control groups by ID number using the Random Integers option at the website [ 11 ]. After recruitment, participants were assigned an ID by the staff member in human resources. These staff assigned participants to groups based on the randomized IDs provided to them. Random allocation occurred only after all participants within a place of employment were recruited and assigned ID numbers. The instructor (Kitchener) provided the human resources staff member with the names of attendees to check that participation was as allocated. Blinding was not possible with the Mental Health First Aid intervention. Ethics Ethical approval for the study was given by the Australian National University Human Research Ethics Committee. Statistical methods Repeated measures analysis of variance was used to analyze continuous measures, with two groups (intervention and control) and two time points (pre-test and follow-up). The principal interest was in the group × time interaction effect. Logistic regression was used to analyze change in dichotomous measures, with group and pre-test score as the predictors and follow-up score as the outcome. Place of employment was also investigated to see if there was a difference in the effects of training. However, no interaction effects involving place of employment were found, so this variable was dropped from all analyses reported below. The analysis was carried out according to intention-to-treat principles, so that all persons who completed a pre-test questionnaire were included, even if they subsequently dropped out. In such cases, the pre-test score was substituted for the missing value, so that no improvement was assumed. Results Recruitment An email inviting participation was sent to all staff of the relevant departments based in Canberra. The email was sent out in May 2002 for the Department of Health and Ageing and March 2003 for the Department of Family and Community Services. In order to participate, staff had to send back a consent form and fill out a pre-test questionnaire before the start of classes. Participant flow Figure 1 shows the flow of participants at each stage of the trial. There were two deviations from plan. Firstly, 18 of the 146 participants (12.3%) assigned to receive Mental Health First Aid training did not complete the whole course. Secondly, 39 out of 146 participants (26.7%) in the intervention group did not complete follow-up questionnaires, compared to only 22 out of 155 (14.2%) in the control group. Figure 1 Flow diagram showing progress through the phases of the trial. Participants' characteristics In terms of sociodemographic characteristics, 78.1% of the participants were female, 49.2% were aged 18–39 years, 50.2% were aged 40–59 and 0.7% aged 60+ years. There were 60.6% with a university degree, 1.3% were aboriginal and 8.6% did not have English as their first language. 13.0% described themselves as mental health consumers, 9.6% as carers for a person with a mental health problem, and 6.3% as health service providers. When asked their reason for doing the course, 27.2% cited reasons relating to their workplace, 11.7% reasons relating to family or close friends, 4.9% reasons relating to their own mental health status, 20.5% cited duty as a citizen, 29% said they were just interested, and 6.7% wanted more accurate or updated information on mental health. 165 (54.8%) of the participants worked at the Department of Health and Ageing and 136 (45.2%) at the Department of Family and Community Services. Numbers analyzed The data were analyzed according to intention-to-treat principles, so that all persons who completed a pre-test questionnaire were included, even if they subsequently dropped out. For every analysis, there were 146 participants analyzed in the intervention group and 155 in the control group. Perception of mental health problem in self or family Participants were asked about whether they themselves had ever experienced a mental health problem or whether anyone in their family had. Table 1 shows that around half reported having personally experienced a mental health problem and around three-quarters reported that a family member had a mental health problem. However, participating in the Mental Health First Aid course did not affect these variables. Table 1 Percent reporting history of mental health problem in self or family. Mental health problems in: MHFA group Control group P-value for group × time interaction Self .577 Pre-test 60.0% 49.7% Follow-up 65.5% 55.6% Change (95% CI) 5.5% (0.5 to 10.6) 5.9% (0.6 to 11.1) Family .849 Pre-test 74.5% 73.0% Follow-up 77.2% 75.7% Change (95% CI) 2.8% (-3.9 to 9.4) 2.6% (-3.5 to 8.7) Recognition of disorder in vignette Table 2 shows the percentage who correctly recognized the disorders in the vignettes. For the schizophrenia vignette, mention of either "schizophrenia" or "psychosis" was considered correct. The table also shows the percentage who got both vignettes correct. Although there tended to be greater improvement in recognition in the group receiving Mental Health First Aid, there were no significant differences from the control group. Table 2 Percent correctly recognizing the disorder in a vignette. Type of vignette MHFA group Control group P-value for group × time interaction Depression .091 Pre-test 90.2% 87.7% Follow-up 95.8% 90.3% Change (95% CI) 5.6% (0.5 to 10.7) 2.6% (-2.8 to 8.0) Schizophrenia .083 Pre-test 74.6% 83.9% Follow-up 82.6% 81.9% Change (95% CI) 8.0% (1.5 to 14.4) -2.0% (-6.8 to 2.8) Both vignettes .189 Pre-test 70.6% 76.5% Follow-up 80.2% 77.8% Change (95% CI) 9.6% (2.8 to 16.4) 1.3% (-5.2 to 7.9) Beliefs about treatments Table 3 shows the data on whether beliefs about treatments became more concordant with those of health professionals. There was significantly greater improvement in concordance in the Mental Health First Aid group when both depression and schizophrenia were considered together. However, the trends failed to reach significance at the .05 level when the disorders were considered separately. Table 3 Changes in beliefs about treatment and in social distance. Scale MHFA group Control group P-value for group × time interaction Beliefs about treatment for depression .062 Pre-test mean (SD) 82.10 (17.27) 83.00 (18.95) Follow-up mean (SD) 86.29 (18.30) 83.42 (18.48) Change (95% CI) 4.19 (1.18 to 7.20) 0.42 (-2.20 to 3.04) Beliefs about treatment for schizophrenia .096 Pre-test mean (SD) 84.28 (19.33) 88.21 (16.76) Follow-up mean (SD) 87.41 (18.26) 88.41 (16.11) Change (95% CI) 3.13 (0.30 to 5.96) 0.20 (-1.87 to 2.27) Beliefs about treatment for both disorders .036 Pre-test mean (SD) 83.28 (16.65) 85.51 (15.05) Follow-up mean (SD) 86.98 (16.78) 85.89 (14.42) Change (95% CI) 3.70 (1.16 to 6.24) 0.38 (-1.46 to 2.23) Social distance from person with depression .005 Pre-test mean (SD) 8.74 (2.80) 8.63 (2.63) Follow-up mean (SD) 7.86 (2.50) 8.46 (2.54) Change (95% CI) -0.88 (-1.23 to -0.53) -0.18 (-0.51 to 0.16) Social distance from person with schizophrenia .211 Pre-test mean (SD) 12.12 (3.53) 12.13 (3.50) Follow-up mean (SD) 11.27 (3.50) 11.62 (3.35) Change (95% CI) -0.84 (-1.23 to -0.46) -0.51 (-0.87 to -0.15) Social distance from both .020 Pre-test mean (SD) 20.88 (5.79) 20.79 (5.53) Follow-up mean (SD) 19.14 (5.43) 20.07 (5.30) Change (95% CI) -1.73 (-2.37 to -1.10) -0.72 (-1.29 to -0.14) Social distance Table 3 shows data on social distance from the person in each vignette. There was greater improvement in social distance in the Mental Health First Aid group overall, but when the two vignettes were examined separately, this improvement was confined to the depression vignette. Help provided to others Table 4 shows data on confidence in providing help and actual help provided to others in the period before completing the questionnaire. Confidence improved more in the Mental Health First Aid group. There was no change in the percentage who reported contact with anyone with a mental health problem or in the percentage reporting giving "some" or "a lot" of help. However, while the control group showed a decline in the percentage advising professional help, the Mental Health First Aid group did not, leading to a significant difference between groups. Table 4 Changes in confidence and help provided to others. Outcome MHFA group Control group P-value for group × time interaction % Feeling confident in helping someone ("moderately", "quite a lot" or "extremely") .001 Pre-test 54.5% 49.7% Follow-up 74.5% 57.4% Change (95% CI) 20.0% (12.6 to 27.4) 7.7% (1.3 to 14.1) % Had contact with anyone with mental health problem .157 Pre-test 71.5% 70.8% Follow-up 72.9% 65.6% Change (95% CI) 1.4% (-6.9 to 9.6) -5.2% (-13.5 to 3.1) % Provided help ("some" or "a lot") .525 Pre-test 37.0% 37.5% Follow-up 39.0% 36.2% Change (95% CI) 2.0% (-5.5 to 9.6) -1.3% (-9.6 to 6.9) % Advised professional help .007 Pre-test 28.1% 27.1% Follow-up 29.4% 16.8% Change (95% CI) 1.4% (-6.8 to 9.5) -10.3% (-18.0 to -2.6) Participants' mental health Table 5 shows changes in the mental and physical health of participants. The Mental Health First Aid group showed significantly greater improvement in mental health. No difference between groups was found in physical health, but none was expected. The physical health scale is included in the table only to show the specificity of the effect on mental health. Table 5 Changes in mental and physical health. Scale MHFA group Control group P-value for group × time interaction Mental health .035 Pre-test mean (SD) 45.43 (11.40) 45.40 (10.17) Follow-up mean (SD) 47.48 (11.11) 45.11 (11.25) Change (95% CI) 2.06 (0.39 to 3.72) -0.29 (-1.72 to 1.14) Physical health .506 Pre-test mean (SD) 51.38 (7.97) 51.97 (8.11) Follow-up mean (SD) 50.74 (8.14) 51.90 (8.68) Change (95% CI) -0.64 (-1.80 to 0.53) -0.07 (-1.29 to 1.16) Adverse events Given that an educational intervention was evaluated with a non-clinical sample, there was no justification for a systematic inquiry into adverse events. Informally, no adverse events were reported. Discussion This trial has found a number of benefits from Mental Health First Aid training. Relative to the control group, the intervention group showed greater confidence in providing help to others, greater likelihood of advising people to seek professional help, improved concordance with health professionals in beliefs about treatment, decreased social distance from people suffering from depression, and improved mental health of the participants themselves. Recognition of disorders in vignettes did not improve, but there was a very high recognition at pre-test, limiting the scope for improvement. A potential criticism of Mental Health First Aid training is that it will lead to excessive labeling of life problems as mental disorders by members of the public. To check this possibility we asked participants about mental health problems in themselves and family members. Although a high prevalence rate was reported, we found that the course had no effect on these rates. A surprising effect was that the course improved the participants' scores on the SF-12 mental health scale. We included this scale to explore whether there was any impact on mental health, but did not have any strong expectation that it would. The course is not aimed at the participants' own mental health and does not include any therapy. Furthermore, only 5% of participants cited their own mental health as a reason for doing the course. Nevertheless, the participants' mean score on the mental health scale was around half a standard deviation below Australian population norms [ 9 ], showing that some were having on-going problems. The cause of the improvement in mental health is not clear. It is unlikely to be a placebo effect because the course gave no expectation of personal change in mental health and only a small percentage did the course for their own benefit. Furthermore, there was no corresponding change on the SF-12 physical health scale. We speculate that the evidence-based information given in the course allowed participants to take action to benefit their own mental health. A similar therapeutic effect has recently been reported from a trial of a web site giving evidence-based information on depression [ 12 ]. The data analysis involved a conservative intention-to-treat strategy in which participants who failed to complete the whole course were included and those who failed to respond to the follow-up questionnaire were assumed to show no change. A particular limitation in the present study is that participants in the intervention group showed a poorer response to the follow-up questionnaire than controls. The reason for this poorer response is unknown, but we believe it occurred because the intervention group had already received the course and had nothing to gain by filling out a further questionnaire. By contrast, the controls were still waiting to receive their training and may have believed that filling out the questionnaire would assist this. Whatever the reason, the poorer response in the intervention group meant that more of them were assumed to show no change, thus minimizing any benefits of the training. It is likely that the true effects of Mental Health First Aid training are greater than the present data indicate. The present trial evaluates efficacy rather than effectiveness. The trial was carried out in a workplace setting with well-educated employees who were allowed to do the course during working hours. There was only one instructor, who was the developer of the Mental Health First Aid course, limiting the generalizability of the findings to other instructors. Further research is needed to evaluate the course as taught by other instructors in more typical settings. We are currently engaged in an effectiveness trial with members of the public in a large rural area, with local health service staff trained to run the courses. The Mental Health First Aid training evaluated in this trial was 9 hours long. Based on feedback from participants that the course needed to be longer, we now routinely run the course over 12 hours. This longer course expands on each of the topics covered, especially substance use disorders. Whether this longer course has additional benefits remains to be evaluated. However, our expectation is that it would produce greater effects on beliefs about treatment, confidence in providing help and actual help to provided to others. Conclusions Mental Health First Aid training appears to be effective in improving some aspects of mental health literacy, confidence in providing help to others, and the type of help provided. The training also benefits the mental health of participants. The course is highly acceptable in a workplace setting and could be widely applied. Over 100 Mental Health First Aid instructors have now been trained and the course is available throughout much of Australia and in Scotland, Hong Kong and New York State, USA. Dissemination in other localities is planned in the near future. Competing interests The authors were the developers of the Mental Health First Aid course. Authors' contributions BAK co-designed the study and the evaluation questionnaire, taught the Mental Health First Aid courses, and co-wrote the manuscript. AFJ co-designed the study and the evaluation questionnaire, analyzed the data, and co-wrote the manuscript. Both authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Pre-test questionnaire. questions used in pre-test questionnaire Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514553.xml
528731	Content-rich biological network constructed by mining PubMed abstracts	Background The integration of the rapidly expanding corpus of information about the genome, transcriptome, and proteome, engendered by powerful technological advances, such as microarrays, and the availability of genomic sequence from multiple species, challenges the grasp and comprehension of the scientific community. Despite the existence of text-mining methods that identify biological relationships based on the textual co-occurrence of gene/protein terms or similarities in abstract texts, knowledge of the underlying molecular connections on a large scale, which is prerequisite to understanding novel biological processes, lags far behind the accumulation of data. While computationally efficient, the co-occurrence-based approaches fail to characterize (e.g., inhibition or stimulation, directionality) biological interactions. Programs with natural language processing (NLP) capability have been created to address these limitations, however, they are in general not readily accessible to the public. Results We present a NLP-based text-mining approach, Chilibot, which constructs content-rich relationship networks among biological concepts, genes, proteins, or drugs. Amongst its features, suggestions for new hypotheses can be generated. Lastly, we provide evidence that the connectivity of molecular networks extracted from the biological literature follows the power-law distribution, indicating scale-free topologies consistent with the results of previous experimental analyses. Conclusions Chilibot distills scientific relationships from knowledge available throughout a wide range of biological domains and presents these in a content-rich graphical format, thus integrating general biomedical knowledge with the specialized knowledge and interests of the user. Chilibot can be accessed free of charge to academic users.	Background A comprehensive understanding of the rapidly expanding corpus of information about the genome, transcriptome, and proteome at large scale requires extensive integration with existing knowledge that often pertains to a number of biological disciplines. Despite the existence of specialized databases (e.g. [ 1 , 2 ]), most of this knowledge is still stored in the form of unstructured free-texts. Different approaches have been developed that automatically retrieve information on molecular interactions from the biomedical literature. Some assume that the co-occurrence of gene/protein names in texts corresponds to a biological relationship [ 3 , 4 ]. Others assign relationships based on similarities in the texts of abstracts [ 5 - 7 ]. While computationally efficient, these methods do not characterize each interaction (e.g., inhibition versus stimulation, directionality). Furthermore, relationships are supported by minimal documentation, other than PubMed IDs. Natural language processing (NLP) has also been used as the basis of programs designed to retrieve more detailed information about molecular relationships ([ 8 - 11 ], reviewed in [ 12 , 13 ]). However, many of these programs were built for testing purposes and are not available to the scientific community at large [ 14 ]. Herein, we present a text mining approach, Chilibot ( ch ip li terature ro bot ), which constructs content-rich relationship networks between genes, proteins, drugs and biological concepts (figure 1 ) based on linguistic analysis of relevant records stored in the PubMed literature database. The nature of each relationship (e.g. inhibitory versus stimulative) is encoded in the network map. The network map is also annotated by sentences describing the relationships (content of the network). For example, there are an average of 24 sentences describing each relationship and 11 sentences describing each query term when a maximum of 30 abstracts are analyzed for each relationship. Thus, Chilibot provides a flexible tool for integrating the rapidly expanding body of biomedical knowledge with the highly specialized knowledge of the individual user. Recent analyses of several types of biological networks (e.g. metabolic [ 15 ], proteomic [ 16 ], and transcriptomic [ 17 ] networks) have found that their connectivities followed the power-law distribution, specifying that the probability of any node connecting to "k" other nodes is proportional to 1/k n . These networks are classified as scale-free networks and are in direct contrast to the bell-shaped distributions seen in random networks [ 15 ]. Since most nodes in a scale-free network have very few connections, yet a few nodes (i.e., hubs) have a large number of connections, scale-free networks are robust, resisting the random failure of nodes, but vulnerable if hubs fail. To facilitate comparisons to the structure of other biological networks, the connectivity of networks constructed by Chilibot were analyzed and found to follow the power-law distribution characteristic of scale-free topologies. Results and discussion Design and implementation The overall goal of Chilibot is to generate graphical representations of the relationships among user provided terms (e.g. molecules, concepts, etc). This is achieved by automatically querying the PubMed literature database and extracting information using natural language processing (NLP) techniques. Chilibot is an Internet-based application [ 18 ]. The system has been tested on FreeBSD and Red Hat Linux operating systems. Users interact with the Chilibot server from web-browsers (e.g. Mozilla Firefox, Netscape, or Microsoft Internet Explorer). Batch queries can also be conducted, but only from the server side. Terms that can be queried include gene symbols, UniGene identifications (including human, rat and mouse) and/or free-form keywords (e.g. "ischemia", "apoptosis", "methylation"). Chilibot retrieves the synonyms of the queried terms from an internal database. The synonym table is compiled from 6 genomic or proteomic databases (see table 1 ). A total of 113,503 unique symbols were collected; amongst these, 62,178 (54.8%) contained at least one alias (figure. 2 ). The synonyms can be edited by users if necessary. Pair-wise queries incorporating the synonyms then are sent to PubMed using the Esearch utility, followed by retrieving relevant records using the Efetch utility. By default, a maximum of 30 abstracts per query are retrieved for analysis, however options are available to retrieve 20–50 abstracts. Both utilities are available from the National Center for Biotechnology Information (NCBI). The texts (including each title and abstract) are then parsed into units of one sentence, which has been shown to yield higher performance levels than paragraphs or phrases in the identification of relationships from MEDLINE abstracts [ 19 ]. Sentences containing both query terms or their synonyms are subjected to part-of-speech (POS) tagging using the TnT tagger [ 20 ], which is followed by shallow parsing using CASS [ 21 ]. A set of rules (see Methods) is followed to classify these sentences into one of five categories: stimulatory (interactive), inhibitory (interactive), neutral (interactive), parallel (non-interactive) and abstract co-occurrence only. The overall relationship between each pair of query terms is then specified based on the relationships found in the sentences (see Methods). Retrieved relationships are visualized using AiSee (AbsInt, Angewandte Informatik GmbH, Germany). Nodes (boxes) are used to represent query terms and lines for relationships. Icons with different shapes and colors are added to the middle of each line to indicate the nature of the relationship, with arrows indicating directionality. Color coding of individual nodes can be used to report the magnitude of change in experimental data, when provided by the user; different shades of green or red represent up- or down-regulation, respectively, and more saturated colors are associated with larger changes. The weight of an interactive relationship, reflecting the number of abstracts obtained from PubMed, is displayed within the icon (figure. 1 ). The co-ordinates of the graphical elements are used to link the documentation of the relationships and the query terms to the map. Typically, querying a list of 10 terms takes 3–4 minutes, allowing 3 seconds between PubMed connections as requested by NCBI. Performance evaluation We used a set of 770 known relationships (see Methods) specified in the Database of Interacting Proteins (DIP) [ 2 ] to measure the performance of Chilibot in finding relationships. DIP was chosen for this purpose because it contains a large number of protein interaction relationships that are manually curated. We defined recall as the fraction of relevant relationships retrieved. The effect of the number of documents analyzed on recall is first evaluated by analyzing a maximum of 5, 10, 20, 30, 40, and 50 of the most recent abstracts for each pair of proteins. Figure 3a shows that analyzing 5 or 50 abstracts achieved recalls of 90.1% and 91.2%, respectively. Thus, increasing the number of documents analyzed does not yield an increase in recall. However, analyzing more abstracts increased the average number of statements demonstrating the relationships (figure. 3a ), resulting in a steady increase in stimulatory or inhibitory relationships and a decrease in "parallel relationships" (figure. 3b ). In addition, we also evaluated the effect of the total number of abstracts available in PubMed on recall. Of the 770 queries conducted by Chilibot, 66 had no reference in PubMed and no relationship was detected. Chilibot also failed to detect a relationship from two queries where each had 1 reference available. Relationships were detected among the 702 remaining queries; the number of references in PubMed ranged from less than 10 (206 queries), between 10 to 99 (299), to more than 100 (197). Thus, the ability of Chilibot to detect relationships depends on the existence of PubMed records, but is not sensitive to the number of references. Chilibot's recall proficiency may be attributable to a large dictionary of synonyms (currently from 6 databases), optimized PubMed query structure and non-alphanumeric character processing method (see Methods), and to the use of both sentences and abstracts as units of analysis. However, we were not able to directly compare the performance of Chilibot with other NLP-based PubMed-mining software because none of these are available to the public [ 14 ]. A potential approach to facilitate such performance comparisons would entail coding software according to published algorithms. However, this is not likely to replicate all details of the original software; thus, the comparisons obtained via such an approach would not be valid. Amongst the 68 DIP relationships that Chilibot did not detect (table 2 ), the largest number represented a failure to recognize abstracts containing generalized protein names (e.g. PKA in PubMed abstract vs. type II-alpha form of PKA in DIP), a limitation also reported for FlyBase [ 22 ]. Recall was also limited by synonym coverage and by the presence of information in the main text, but not in the abstract. Since many of the DIP relationships were originally based on the main text of a single reference [ 2 ], the high recall of Chilibot depends on the redundancy of information in the literature. To estimate precision, defined as the fraction of retrieved relationships that are relevant, we randomly selected 100 relationships from the 702 relationships recovered by Chilibot (86 interactive, 11 parallel, and 3 abstract co-occurrence). We manually confirmed that the documentation retrieved by Chilibot contained information about 96 of the targeted relationships, and the remaining four shared symbols with other genes. In the interactive category, directionality was correctly identified in 79.1% and inhibitory/stimulatory properties in 74.4%. The original data used to perform these analyses are available [see additional file 1 and 2 ]. User interface features One of the key features of Chilibot is its capacity to link the relationships represented in the network map directly to their supporting documentation, usually as sentences containing both of the query terms. In addition, each node is linked to its synonym list and to a set of statements demonstrating the use of the term; these statements are selected from abstract texts by an algorithm favoring conclusive statements (see Methods). By providing the literature in a condensed and highlighted form, Chilibot facilitates the rapid comprehension of the relationships by the user. Chilibot provides several options for customizing the query process and for viewing the identified relationships. Context specific searches restrict the analysis of relationships to a specific subject area, as defined by the user. Internet searches can also be customized (e.g. searching only documents in PDF format) by using Google WebAPI. Specific subsets of relationships contained in an overall relationship map can be reconfigured. For example, the user can customize the relationship map by requesting only those relationships with direct linkage to a specific node, or those that have a requisite number of supporting publications [see additional file 3 and 4 for examples]. Chilibot also identifies key index terms common to the relationship network. To do so, Chilibot uses Medical Subject Headings (MESH) [ 23 ], a controlled vocabulary that indexes the subjects of the documents developed by the National Library of Medicine. Chilibot ranks MESH keywords indexed in the literature that supports the relationship network. The ranking is determined by the frequencies of the keywords, as well as whether the keyword is a major or minor topic of the paper (see Methods). The top ranked keywords, reflecting the subject area(s) shared by the query terms, can serve as a guide for further reading and suggest new Chilibot queries. Chilibot also has the capability of suggesting new hypotheses based on the retrieved network of relationships. Such hypotheses, originally described by Swanson et al. as "undiscovered public knowledge" [ 24 ], referred to the inference of an interaction between two items A and C, based on knowledge that A affects B and B affects C. This involves software that generates a large list of "B" terms from titles returned by PubMed queries. The user filters these terms, aided by the titles and abstracts. Variations of this method have been designed and tested by others [ 25 , 26 ]. Taking a similar approach, Chilibot scans the network of retrieved relationships to find pairs of nodes that have no documented relationship, but have connections to a common tertiary node(s). These pairs of nodes are classified as having a "hypothetical relationship". The networks that contain these "hypothetical relationships", including the tertiary node(s), are then provided to the user in graphical format, with links to their documentation. To test the value of these "hypothetical relationships" in predicting the results of future research, we queried 22 genes known to be involved in long-term potentiation (LTP), an electrophysiological phenomenon closely associated with memory formation. Chilibot identified a direct relationship between LTP and all 22 genes, along with 194 inter relationships amongst the 22 genes. We then performed retrospective studies by limiting the search to literature published before the years 2000, 1995 and 1990 [ additional file 5 contains all the original search results]. The LTP-related "hypothetical relationships" identified by Chilibot, using these date-limited reference sets, are listed in table 3 . As an example, by 1990, the involvement of calcium calmodulin kinase type II (CaMKII) in the induction of LTP had been established [ 27 ]. It was also known that CaMKII phosphorylates synapsin I [ 28 , 29 ]. Based on these and similar relationships (see table 3 ) that were documented in the literature available by 1990, Chilibot predicted the involvement of synapsin I in LTP, which was subsequently demonstrated empirically by 1995 [ 30 ]. Retrospective analyses like these depend on the progression of specific knowledge in scientific fields during a particular time period. Thus, if we were to test a different set of search terms, we would not expect to obtain the same number of suggested hypotheses, nor would we expect the same proportion of such hypotheses to be validated by the current literature. Based on the literature that is currently available, Chilibot identified new hypothetical relationships, such as those between synaptophysin/CREB and synaptotagmin/CREB. Currently no direct empirical evidence for these relationships is available. However, scanning the 5' untranslated region of the synaptophysin and synaptotagmin genes did show multiple CREB binding sites, providing bioinformatics-based evidence supporting the plausibility of these potential interactions. Although these examples are promising, they are hypothetical relationships. Further review of the scientific literature, such as the sentences provided by Chilibot, is required to clarify the rationale for these hypotheses. Network topology of relationships retrieved from the literature Recent large-scale studies of metabolic [ 15 ], transcriptomic [ 17 ] and proteomic [ 16 , 31 ] networks, based on analyses of experimental data, have found that their topologies belong within the class of scale-free networks. For comparison to the preceding biological networks, we studied the connectivity of the literature-based networks obtained by applying Chilibot to three groups of randomly selected genes (300 genes per group). The resulting networks contain 224, 116, and 138 nodes and 3018, 962, and 1912 relationships, respectively. Visualization of the network structure of one of the groups is provided [see additional file 6 ]. The connectivity of the 3 groups was averaged and plotted in figure. 4 , showing a power-law distribution. The relatively low value of n = 1.21 (n is approximately 2 in many of these networks [ 15 , 32 , 33 ]) may reflect the fact that many relationships are yet to be documented. In addition, we also found a positive correlation between the number of abstracts available per node and the number of connections to that node (R 2 = 0.76, p < 0.001). This suggests that the discovery of biological relationships attributable to specific nodes might be influenced both by the amount of scientific effort deliberately devoted to understanding that node and the intrinsic connectivity of that node. Although the commitment of greater resources by the scientific community to certain nodes may bias the topology of the scientific literature to some extent, this is likely to be regulated and limited by the strength of the findings, which would be directly related to the intrinsic connectivity of a particular node. Thus, it is reasonable to postulate that the topology of the biomedical literature on gene/protein interactions may reflect that of the interactions per se . The scale-free topology of gene/protein relationships provides another dimension for comparing and prioritizing research targets after large-scale experiments. Currently, genes or proteins with large-fold changes are generally favored for further study [ 34 ]. However, by itself, a large-fold change may be insufficient to predict whether such molecules are pivotal in the regulation of important biological processes. For example, in many biological signaling pathways, a small increase in up-stream events (such as the binding of a peptide or hormone to its receptor(s)) is usually associated with a hundred to thousand-fold increase in down-stream events [ 35 , 36 ] (e.g., activation of mitogen-activated protein kinases or the production of cAMP). Therefore, knowledge of a network's critical nodes (i.e. hubs), which may be predicted by network connectivity [ 32 ], is likely to increase the power and efficiency of identifying potential experimental targets capable of modifying network function. Conclusion Chilibot graphically summarizes the relationships amongst a large set of user provided terms by analyzing abstracts retrieved from the PubMed literature database. We have found in our benchmark tests that these retrieved relationships are reliable. We believe that the scientific community will benefit from this literature mining capability along with the many features that Chilibot provides, especially in an era of science when insight can be submerged in an overwhelming sea of data and modularized knowledge. Methods Constructing the nomenclature dictionary Flat text file versions of the six databases (HUGO, LocusLink, OMIM, GDB, SwissProt, and SGD) were downloaded from their corresponding ftp sites. Symbol-name pairs were extracted from the corresponding fields using Perl scripts. Names were curated to remove words that are unlikely to be used in texts, such as "partial cDNA", "fragment", etc. In addition, non-alphanumerical characters were converted into spaces. Entries with the same symbol from the six databases were then combined in a case insensitive manner. The final dictionary is stored in the Postgresql relational database. Optimization of PubMed querying method The NCBI Eutilies, in particular Esearch and Efetch, are used in conjunction with the Perl LWP module to interact with the server. Optimization was necessary because phrase or adjacency searches are not supported by PubMed. Thus, when searching for names with multiple words, it is possible to retrieve abstracts that contain all the relevant words, however the words are used in different places of the abstract. Further, PubMed has an automatic term mapping feature that converts user input according to the MESH translation table. For our purposes, we considered this an undesirable feature. After small scale testing, the query structure we selected places a title and abstract restriction tag ([tiab]) after the name of the query term. This disables the term translation feature and also treats the term as a phrase when possible, according to PubMed documentation. To test the effectiveness of this strategy, we sampled 510 names with lengths ranging from 1 to 11 words. A total of 4584 abstracts were retrieved. We were able to find the query name from 4487 (97.9%) of the abstracts. We thus constructed the pair-wise PubMed query in the following format: (Term 1 synonym 1 [tiab] OR Term 1 synonym 2 [tiab] OR ...) AND (Term 2 synonym 1 [tiab] OR Term 2 synonym 2 [tiab] OR ...) Acronym disambiguity Many methods [e.g. [ 37 - 40 ]] have been developed to translate acronyms unambiguously into their full length terminology, since acronyms may have multiple meanings and become a source of false positives [ 3 , 41 ]. Chilibot provides an option to verify the meaning of acronyms when they are used as the query term. When a relevant acronym first appears, Chilibot retains a phrase immediately preceding the acronym that contains the same number of words as the number of characters in the acronym. The phrase then is compared to all synonyms of the acronym, which are retrieved from the nomenclature database of Chilibot. The abstract is excluded from analysis if less than 30% of the words in the phrase are found in the synonym list. Context sensitive search All the context keywords provided by the user are combined with an "OR" operation. This string is then combined with the pair-wise PubMed queries, using an "AND" operation. The context keywords are not used in subsequent analyses. Synopsis generation A synopsis is a collection of sentences used to annotate the query terms. It is generated from the first 100 sentences that contain the specific query term or its synonyms. These sentences are sorted by a weighting mechanism that favors short, conclusive sentences. Words suggesting a conclusion, such as "suggest", "found", "show", "data" etc weights as +9 points. Starting the sentence with the query term and a verb weights as +5 points. The presence of words suggesting a negative result such as "not", "lack", "fail", "without" is weighted as -3 points. Having more than 30 words also reduces the weight by 3 points. Lastly, having keywords specified by the user adds 5 points to the weight. The 15 sentences with the highest weights are displayed. Natural language processing Title and abstract texts retrieved via the Efetch utility are first parsed into individual sentences using a Perl script. Only sentences containing both of the query terms or their synonyms are subjected to NLP analysis, which includes POS tagging by the TnT software [ 20 ] and shallow parsing by the CASS software [ 21 ]. Testing TnT on a small corpus of 10 PubMed abstracts (2646 words), using the supplied WSJ language model, showed 537 (20.29%) unknown words. Manual inspection identified 150 errors in the assigned POS tags. We then trained the TnT software with the GENIA corpus [ 42 ] (a collection of 2000 PubMed abstracts annotated with POS and other information). Re-analyzing the same 2646 words, using the customized language model, resulted in only 289 (10.92%) unknown words. Manual inspection identified 31 errors. Thus, the language model based on the GENIA corpus was used for all subsequent analyses. CASS software was used without further adjustment. Classification of relationships All sentences containing two query terms (or their synonyms) are classified into one of six categories: stimulatory (interactive), inhibitory (interactive), both stimulatory and inhibitory (interactive), neutral (interactive), parallel (non-interactive) and abstract co-occurrence only. Sentences are classified into interactive or non-interactive relationships based on the presence or absence of a verb phrase between the two query terms. The following exceptions apply: sentences are classified as parallel when the query terms are present in two separate clauses; sentences without a verb phrase between the query terms, but with specific terms indicating interactions such as "interaction", "bind", etc., are classified as interactive; interactive relationships are converted into parallel relationship when there is a negation (such as "not") within the same clause of the verb phrase. The interactive relationship is further classified into stimulatory, inhibitory, or neutral subtypes based on the presence or absence of words describing such relationships, including "activate", "facilitate", "increase", "induce", "stimulate", "enhance", "elevate", "inactivate", "abolish", "attenuate", "block", "decrease", "eliminate", "inhibit", "reduce", "suppress". For interactive relationships, the direction is defined as from the left query term to the right term and is reversed when passive voice is detected. To avoid the influence by spurious mistakes, the overall relationship between two terms is defined as interactive only when more than 20% of the sentences are detected as either stimulatory or inhibitory. Lastly, the co-occurrence type is assigned when the two query terms are located in the same abstract but not the same sentence. We ranked the informativeness of the relationships in the following order: both stimulatory and inhibitory, either stimulatory or inhibitory, neutral interactive, parallel, abstract co-occurrence. The overall relationship between two query terms is classified as the most informative type of relationship. Visualization of the networks Network layout is generated using the aiSee software. Each pair of query terms identified as having relationships is specified by nodes and represented by square boxes. The relationships are represented by solid lines. A special node with unique identification (an icon) is inserted into the middle of each line. The icon is either circular or rhomboidal depending on the relationship it represents (see legend of Figure 1 ). The network map as well as the links from the map to the descriptions of the relationships are obtained by calling the command line interface of aiSee. "Hypothetical relationship" generation and testing After the query session is finished, the user can request Chilibot to suggest hypothetical relationships for any node that is within the retrieved network. For each node requested (NR) by the user, Chilibot scans the retrieved network to find those nodes that are not directly linked to NR, but have connections to the same tertiary nodes as NR. Chilibot then produces a new network map for each of these "hypothetical relationships", while maintaining the links to the supporting documentation. To test the usefulness of these "hypothetical relationships" in predicting future research, a total of 22 terms (ACTIN, ACTININ, AMPA, ARC, ATF, CAMKII, CAMKIV, CREB, ERK, KV4.2, NMDA, PI-3K, PKA, PKC, PLC, SYNAPSIN I, SYNAPTOPHYSIN, SYNAPTOTAGMIN, TAU, TRKA, TRKB, AND ZIF268) were queried together with LTP (long-term potentiation). Retrospective studies were performed by querying these terms again while adding the PubMed date limiting tag "&mindate=1960&maxdate=$maxdate", where the $maxdate equals to 1990, 1995, 2000, respectively. MESH themes The MESH Keywords of the abstracts represented by the graph are collected and sorted by their weighted percentage. When the keyword is the major topic of the publication, it is weighted as 3. Otherwise, it is weighted as 1. The weights are then divided by the number of abstracts to obtain the weighted percentage. Web search and content filtering Google WebAPI is accessed through Perl scripts. Due to the limitation of the WebAPI, the query terms are searched directly without the expanded synonyms. The URIs of the top 10 hits were retrieved from Google and then the content of these pages was obtained from their individual servers. These pages are then converted into texts, and sentences containing either one of the query terms are presented to the user. Sentences containing both of the query terms are highlighted. Links are also provided to restrict the web search to educational institutions or to files in the portable document format (PDF). Google is a trademark of Google Technology, Inc. Selection of relationships from the Database of Interacting Proteins (DIP) DIP [ 2 ] is a curated protein interaction database. The version of DIP database released on April 18th, 2003 contains 18494 interactions between 7141 proteins. Relationships that originated from large scale genomic or proteomic studies were excluded, reflecting poor reliability of the data [ 43 ] and the low probability that such interactions would be described in textual forms. Proteins with no SwissProt annotation or of yeast origin were also excluded to further reduce the number of relationships to a manageable subset. This selection procedure resulted in a total of 770 relationships. Authors' contributions HC conceived of the project (together with BMS), coded the Chilibot program, performed the evaluations and drafted the manuscript. BMS conceived of the project (together with HC), participated in its design, coordination and analysis, and edited and revised the manuscript. Note All none-graphic files are archived with tar and compressed with bzip2 to reduce file size. Supplementary Material Additional File 1 A total of 770 known relationships were used to test the recall and precision of Chilibot. A maximum of 5, 10, 20, 30, 40, or 50 most recent PubMed records for each relationship was specified for analysis. The relationships identified by Chilibot are summarized and provided in Microsoft Excel and OpenOffice format. Click here for file Additional File 2 The original results of the above study (non-essential files are deleted to keep the file size under the limit set by BMC bioinformatics). Click here for file Additional File 3 Sub-network graph obtained by filtering figure 1 using the number of supporting publications as a threshold criterion. Click here for file Additional File 4 Sub-network graph obtained by filtering figure 1 to selectively display a node of interest (i.e. "cocaine") and other nodes that directly connected to it. Click here for file Additional File 5 The original Chilibot query results of the term "long-term potentiation (LTP)" and 22 other terms, limiting the latest references analyzed to the years 1990, 1995, 2000, and 2004. Click here for file Additional File 6 A graph demonstrating the scale-free topology of relationship networks derived from the biological literature. The network contains 138 nodes and 1912 relationships. A small fraction of the nodes (10 nodes colored in black) accounted for more than 45% of the relationships (solid lines), a characteristic of scale-free topology. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC528731.xml
521067	Cross-species comparison significantly improves genome-wide prediction of cis-regulatory modules in Drosophila	Background The discovery of cis-regulatory modules in metazoan genomes is crucial for understanding the connection between genes and organism diversity. It is important to quantify how comparative genomics can improve computational detection of such modules. Results We run the Stubb software on the entire D. melanogaster genome, to obtain predictions of modules involved in segmentation of the embryo. Stubb uses a probabilistic model to score sequences for clustering of transcription factor binding sites, and can exploit multiple species data within the same probabilistic framework. The predictions are evaluated using publicly available gene expression data for thousands of genes, after careful manual annotation. We demonstrate that the use of a second genome (D. pseudoobscura) for cross-species comparison significantly improves the prediction accuracy of Stubb, and is a more sensitive approach than intersecting the results of separate runs over the two genomes. The entire list of predictions is made available online. Conclusion Evolutionary conservation of modules serves as a filter to improve their detection in silico . The future availability of additional fruitfly genomes therefore carries the prospect of highly specific genome-wide predictions using Stubb.	Background Several computational approaches to the problem of predicting cis-regulatory modules ('CRM's) have been reported recently. Berman et al . [ 1 ], Markstein et al . [ 2 ] and Halfon et al . [ 3 ] predicted CRM's involved in body patterning in the fly, and experimentally verified their predictions. The underlying principle in these algorithms was to detect dense clusters of binding sites, as determined by matches (above some threshold) to catalogued transcription factor weight matrices. The algorithm of Rajewsky et al . [ 4 ], called Ahab, avoided the use of thresholds on weight matrix matches by a probabilistic modeling of CRM's. Ahab predictions within the segmentation gene network were subjected to extensive experimental validation, with excellent overall success (Schroeder et al . [ 5 ]). Most predicted CRM's, when placed upstream of a reporter gene, faithfully reproduce one or more aspects of the endogenous gene expression pattern. Moreover, an analysis of binding site composition over the entire set of validated modules reveals that Ahab's prediction of binding sites correlates well with expression patterns produced by the modules and suggests basic rules governing module composition. The Stubb algorithm (Sinha et al . [ 6 ]) extended Ahab's approach by incorporating the use of two-species sequence information. Stubb also allows the option of scoring positional correlations between binding sites, but this option was not exercised in this study. For each sequence window analyzed, Stubb first computes the homologous sequence in the second species and aligns them using LAGAN (Brudno et al . [ 7 ]). The sequence is then partitioned into "blocks" (contiguous ungapped aligned regions of high percent identity) and non-blocks (sequence fragments between consecutive blocks, in either species). Putative binding sites in blocks are scored under an assumption of common evolutionary descent, using a probabilistic model of binding site evolution. Thus a "weak" site that is well conserved will score higher, while a "strong" site that is poorly conserved will have its score down-weighted. The score of the sequence window includes contributions from binding sites in blocks as well as in non-blocks. Stubb is implemented so that it can be run either on single species or two species data. In the single species mode, it is practically identical to the Ahab program. The Stubb software is available for download from In this paper, we present evidence that the exploitation of cross-species comparison (between D. melanogaster and D. pseudoobscura ) using Stubb can lead to a significant improvement in the accuracy of genome-wide CRM prediction. To our knowledge, this is the first direct evaluation of the effect of cross-species comparison on CRM prediction on a genome-wide scale. Another important contribution of this paper is to present a benchmark for evaluating genome-wide CRM prediction tools, collected from the BDGP database and the literature, and curated by manual inspection of several hundred expression patterns. Using the same benchmark, we evaluate the effect of varying how background sequence information is incorporated in the algorithm, since this is the only tunable parameter in the Stubb program, other than the module length. We are thus able to suggest the optimal parameter settings for genome-wide CRM prediction using Stubb. Finally, we report all genome-wide predictions for cis-regulatory modules involved in anterior-posterior patterning in the early fly embryo, using both single-species and two-species Stubb, many of which make a strong case for experimental validation. Segmentation gene network The transcription control paradigm we use as our test system is the segmentation of the anterior-posterior (ap) axis during early Drosophila embryogenesis, which has long been one of the preferred arenas for studying transcription control in vivo . The segmentation genes form a hierarchical network that, in a process of stepwise refinement, translates broad, overlapping expression gradients into periodic patterns of 14 discrete stripes, which prefigure the 14 segments of the larva (for reviews see St Johnston & Nusslein-Volhard [ 8 ]; Rivera-Pomar & Jackle [ 9 ]; Furriols & Casanova [ 10 ]). The maternal factors form gradients stretching along the entire ap axis of the embryo, the zygotic "gap" factors are expressed in one or more broad slightly overlapping domains; together they generate the 7-stripe patterns of the pair-rule genes; finally, the segment-polarity genes are expressed in 14 stripes. The regulation within the segmentation gene hierarchy is almost entirely transcriptional, and most of the participating genes are transcription factors themselves, activating (in the case of the maternal factors) or repressing (most gap factors) the transcription of genes at the same level or below. In most cases, the relevant binding sites are clustered within a small interval of 0.5–1 kb; these CRM's typically contain binding sites for multiple transcription factors and multiple binding sites for each factor. The clustering and the combinatorial and redundant nature of the input facilitate the computational search for segmentation control elements. Since the expression patterns of the segmentation genes are typically complex, their control regions often contain multiple separate CRM's controlling different aspects of the pattern. The segmentation paradigm has been used as a test system for the computational detection of CRMs by us and others (Rajewsky et al . [ 4 ], Schroeder et al . [ 5 ], Berman et al . [ 1 ], Grad et al . [ 11 ]). Here, as before (Schroeder et al . [ 5 ]), we use the maternal and zygotic gap factors Bicoid, Hunchback, Caudal, Knirps, Krüppel, Giant, Tailless, Dstat, and the TorRE binding factor as input to Stubb. The binding site specificity of each factor is characterized by a position weight matrix that is based on a collection of experimentally verified binding sites. Evaluation methodology The complete genomes of two fruitflies, D. melanogaster and D. pseudobscura have been sequenced, and Stubb was used to predict CRM's in the D. melanogaster genome. This was done in two modes – (i) STUBBSS, where Stubb is run on D. melanogaster genomic sequence alone, and (ii) STUBBMS, where Stubb uses orthologous sequence data from D. pseudobscura to help predict CRM's in D. melanogaster . For each mode of execution, we obtain a separate list of predicted CRM's, sorted in order of confidence in the prediction. The ideal test for our purpose would be to compare the accuracy of these two sorted lists. However, the set of experimentally verified CRM's involved in this system is sparse compared to the size of the system – roughly 50 CRM's are known (including the 15 new modules from Schroeder et al . [ 5 ]), while the number of target genes is several hundreds, by our estimate. Hence, direct evaluation of the success-rate of predictions is not feasible, and we use an alternative source of information to evaluate predictions, as described next. A functional CRM directs the expression of a gene, by definition, and typically this gene is located in close proximity to the CRM. Hence, we may map the list of predicted CRM's to a list of predicted blastoderm-patterned genes – for each CRM predicted by Stubb, the nearest gene is identified, and if this gene is less than a threshold distance of 20 Kbp away, it is predicted to be a blastoderm-patterned gene. The resulting list of predicted "patterned genes" may now be evaluated for accuracy. (Any duplicates in the list are removed before evaluation.) The Berkeley Drosophila Genome Project (BDGP) has catalogued the expression patterns of a large number of genes in D. melanogaster , at various stages of development. We considered such a catalogue of 2167 genes, obtained from BDGP and from the literature. (See Test Genes [ Additional File 1 ].) Visual inspection of the expression patterns of these genes revealed that 286 of them can be classified as having patterned expression along the anterior-posterior axis. (See Materials and Methods; also Patterned Genes [ Additional File 2 ].) Hence, our benchmark is the entire set of 2167 genes, the "positive" set is the 286 ap-patterned genes, the remaining 1881 forming the "negative" set. This enables us to evaluate the accuracy of lists of patterned genes predicted by STUBBSS and STUBBMS, and compare their performance. We note that some accuracy is lost in the translation of a list of predicted CRM's to the predicted genes it is mapped to, as per the mapping defined above. For instance, it is known that CRM's may control a gene located at large distances, i.e., further than the distance threshold of 20 Kb used in the mapping procedure. Also, it is possible that a CRM is located close to two genes, and directs the expression of both genes, or only of the farther gene, being somehow insulated from the nearer one. To address these concerns, we repeat our evaluation with a slightly different mapping from the one described above. A caveat that remains is that there may be genomic sequences that are functional, in the sense that they are capable of directing a specific blastoderm pattern in reporter gene constructs, but whose activity is 'silenced' in native genomic context and does not translate to patterning of any gene. Also, the CRM may direct expression of the gene only at post-blastodermal stages, so that the gene is not included in the "positive" test set of blastoderm patterned genes. Conversely, it may also happen that a predicted CRM lies close to a patterned gene, thereby being counted as a true positive, but the predicted CRM is not the sequence responsible for the gene's regulation. We assume that such effects are not biased against either algorithm. Results STUBBMS performs significantly better than STUBBSS Figure 1a shows the results of our evaluation procedure on STUBBSS and STUBBMS. These results are for the best choice of parameters for each algorithm – local, 1 st order background for STUBBSS and global, 2 nd order background for STUBBMS. (The meanings of these parameter values are explained later in this section.) The x-axis is the number of unique genes that are predicted by the algorithm (by progressively decreasing its score threshold) and are in the set of 2167 genes with expression information. On the y-axis we plot how many of those predicted genes are in the "positive" set (i.e., have an ap blastoderm pattern.) Thus, the y-axis is the specificity of the algorithm. We observe that STUBBMS performs significantly better than STUBBSS. For instance, to predict 100 genes correctly, STUBBSS has to make 343 predictions, while STUBBMS only has to make 267 predictions. Figure 1b plots the difference in the number of correct predictions as a fraction of the number of correct STUBBSS predictions, i.e., the percentage change in specificity for the same number of predictions made by either algorithm. We find a typical improvement of over 20%, even when over 300 overall predictions are made by each algorithm. Figure 1c shows the progression of each algorithm's prediction specificity in a moving window of 50 predictions. We find that STUBBMS has a significantly higher hit rate for the first ~120 predictions, after which both algorithms perform comparably. Even for the lower ranked predictions (i.e., those below rank 120), we find a specificity of 20 – 35% with STUBBMS, which is roughly twice the random expectation of 13% based on 286 positives in 2167 genes. In order to further scrutinize the difference in predictions made by the two modes of Stubb, we focused on the points where their difference is most pronounced. Thus, in the top 102 unique gene predictions (for which we have information), STUBBSS reports 39 positives, while STUBBMS scores 61 hits, an improvement of over 56%. In comparison, the random expectation is ~13.5 hits. Thus the predictions of both STUBBSS and STUBBMS are significantly enriched in patterned genes (P < 10 -12 and <10 -37 respectively, Binomial Proportions test). Further examination of the top 102 gene predictions made by each algorithm revealed that 24 true positives are common to both lists. STUBBMS reports 37 true positives not discovered by STUBBSS, while the latter reports 15 true positives not found by the former. Similar results are seen for the top 311 predictions (another peak in Figure 1b ): 70 correct predictions were common to both algorithms, 42 were predicted by STUBBMS only, and 21 by STUBBSS only. Thus there is substantial exclusivity in the sets of true positives of each algorithm. We next examined separately the following three sets of genes: (i) INTERSECTION (predicted by both algorithms in the top 311) (ii) MS-ONLY (predicted only by STUBBMS) and (iii) SS-ONLY (predicted only by STUBBSS). Table 1 shows the break-down of these sets in terms of the strength of expression of their member genes. Overall, 124 of the 286 patterned genes, i.e., about 43%, are strongly expressed. We find in Table 1 that the sets INTERSECTION and MS-ONLY have more strongly expressed genes than weak and intermediate ones, and the opposite trend is seen in the set SS-ONLY. One possible strategy that uses two-species sequence is to make predictions using STUBBSS on each of the two genomes separately and then intersect the respective lists. We found this strategy to be very restrictive – for instance, with a particular score threshold, STUBBSS predicts 205 unique genes in D. melanogaster , but intersecting these predictions with a similar number of top predictions in D. pseudoobscura gives only 68 unique genes, 33 of which are patterned. Of the top 68 predictions made in D. melanogaster alone, 29 are patterned. Thus the "intersection" strategy yields only a modest improvement over the single-species search, and does so at the price of significantly reducing the total number of predictions. Similar results were obtained when intersecting modules instead of gene predictions. We have noted above that the evaluation method is influenced by the way we map the predicted CRM's to predicted genes. To offset potential biases induced by this mapping, we repeated our analysis with a slightly different evaluation procedure, borrowing from the approach of Grad et al . [ 11 ]. We now traverse the sorted list of CRM's and count a CRM as a prediction if either of its two flanking genes has expression information. Furthermore, we designate a prediction to be "correct" if either of the two flanking genes has a blastoderm pattern. The assumption, as in Grad et al . [ 11 ], is that any predicted CRM near a blastoderm-patterned gene is a functional CRM responsible for some aspect of the pattern. Also, we are now counting modules rather than genes, i.e. we are allowing for multiple hits to the same gene. Figure 2a plots the results of STUBBSS and STUBBMS as per this new method of counting predictions and hits. We again notice a significant improvement in STUBBMS. For instance, in the top 300 CRM predictions for which a neighboring gene has expression information, STUBBMS makes 160 correct predictions while STUBBSS scores 121 hits. The gap between STUBBSS and STUBBMS increases as more predictions are considered, so that the improvement consistently stays above 20%, as seen in Figure 2b . For the remainder of this section, our evaluation method will use the more stringent mapping described earlier, wherein the nearest gene is predicted. Since our test data is in the form of lists of genes, we adhere to the evaluation strategy that counts genes. It is clear from Figures 1 and 2 that counting modules rather than genes improves the prediction accuracy, due to multiple CRM predictions for some blastoderm-patterned genes. The default mapping from CRM's to genes used in our evaluations predicts a gene to be patterned only if its proximal end is less than 20 Kb from the CRM. Schroeder et al . [ 5 ] studied the range of locations of experimentally verified CRM's relative to the gene. They found that while there is a clustering of CRM's within the proximal 5 Kb region upstream, downstream or intronic of a gene, it is not unusual to have CRM's more than 10 Kb away from the regulated gene. Nelson et al . [ 12 ] observe that for D. melanogaster, the intergenic space on either side of a gene has a mean of 2 Kb – 10 Kb, depending on the complexity of the gene's function. We repeated our evaluation with different values of the distance threshold, and found that lower thresholds (5 Kb, 10 Kb) decrease the recovery rate, while higher thresholds (50 Kb) do not affect performance. (Data not shown.) Genes in the segmentation hierarchy often have multiple aspects to their expression pattern, with more than one CRM regulating them. We therefore measured how the Stubb predictions fare if we required that each predicted gene be evidenced by at least two predicted CRM's. This heuristic improves the performance of STUBBSS more prominently than that of STUBBMS, though much fewer predictions are made by either algorithm. (See Figure 3 .) While 342 unique gene predictions were made by STUBBSS (Figure 1 ), we now observe that only 105 predictions are made using the same score threshold and the new way of counting predictions. Thus, it appears that STUBBSS performance is open to considerable improvement in the top ~100 predictions, by using either the multiple CRM restriction or the second species' sequence data. The two-species strategy however is able to increase specificity without loss of sensitivity. We have, in all tests reported in this paper, used as input a set of 2167 genes whose expression patterns are available either from BDGP or from the literature. BDGP has a supplementary list of 2065 genes for which only textual annotation has been made public, since these genes have been found to be either (i) ubiquitously expressed at all developmental stages, (ii) not expressed at any stage, or (iii) only maternally expressed. (See Additional Genes [ Additional File 3 ].) Inclusion of these supplementary genes in our data set would approximately halve the overall fraction of patterned genes. When we examine the performance curves of STUBBSS and STUBBMS for this pattern-diluted data set (Figure 4 ), we find that STUBBMS shows an improvement over STUBBSS similar in proportion to that in the default data set, even though the prediction specificity of both programs suffers a drop (as compared to that in Figure 1a ), typically in the range of 10–30%. Note, however, that this is substantially lower than the 50% drop one would expect by chance, given that the total number of genes has almost doubled, while the number of patterned genes remains constant. Characteristics of genes predicted by STUBBMS Our annotations of the blastoderm patterned genes also include whether the gene expression is strong, weak or of intermediate strength; if it has a dorsal-ventral (dv) modulation in addition to the primary anterior-posterior pattern; and if the gene belongs to a "core" set of 48 genes that have been shown experimentally to be required for the segmentation of the embryo (Schroeder et al . [ 5 ]). We were therefore able to examine the characteristics of the genes correctly predicted by Stubb, along these axes of information. The top 135 correct (gene) predictions made by STUBBMS were examined progressively, 20 predictions at a time. (That is, the correct predictions ranked I to I+19 were examined, with I being incremented in steps.) In each step, we computed the fraction of the 20 genes that belonged to the following three non-exclusive categories: (i) genes with dv (in addition to ap) modulation, (ii) genes with strong expression pattern, and (iii) genes in the "core" set of 48 genes. These values are reported in Figure 5 . We find that 1. Genes with dorsal-ventral aspects to their blastoderm pattern are more frequent at lower ranks of prediction; i.e., the top predictions are enriched in genes with anterior-posterior patterns only. 2. Core genes are predominantly found in the top predictions. 3. Genes found at higher ranks are somewhat more likely to be strongly expressed. The first two observations imply that the genes more directly involved in the ap axis formation are recovered at better ranks, and that the lower rank genome-wide predictions are richer in derivative patterns characteristic of genes with more complex regulatory inputs (pair-rule factors, dv factors etc.). The same trends were found for the correct predictions made by STUBBSS. (Data not shown.) Optimal parameter settings for Stubb We next evaluate the effect of varying how background sequence information is incorporated in the Stubb algorithm. This is the only configurable aspect of the program, other than the module length. (In a separate test, we ran Stubb with a module length of 700 instead of the default value of 500, and found no significant difference in the prediction specificity curve.) One important parameter is the "Markov order" of background. A value of k for this parameter means that local correlations are assumed to be present at the level of ( k+1 )-mers, i.e., the random probability of seeing a particular base at a position depends on the bases seen at the previous k positions. (For readers familiar with the studies of Rajewsky et al . [ 4 ] and Schroeder et al . [ 5 ], "background k " in those studies is the same as a (k-1) th order background in the terminology of this paper.) We vary this parameter to take the values k = 1 and k = 2 , in different runs. The other parameter is the actual sequence used by Stubb to measure background nucleotide frequencies. Here the two options are (i) to use the current sequence window as background, or (ii) to use a pre-specified sequence (or collection of sequences) as background. We call these two the "local" and "global" background models respectively. For the "global" model, we input into Stubb 150 Kb of sequence from non-coding regions of the D. melanogaster genome, collected from the five chromosome arms 2L, 2R, 3L, 3R, and X. Figure 6 plots the specificity curves (as in Figure 1a ) for each combination of parameter values tested. Figure 6a reports on different variants of STUBBSS, while Figure 6b plots the performance of STUBBMS. We find that STUBBSS performs best with a local, 1 st order background, though the other parameter values produce only slightly different results. On the other hand, the effect of background parameters on two-species Stubb is more pronounced, with the best choice being a global, 2 nd order background. Using a global 1 st background order gives almost identical results (data not shown), hence we infer that a global background is the optimal choice for STUBBMS. As mentioned earlier, the STUBBSS program implements the same class of algorithm as the Ahab algorithm of Rajewsky et al . [ 4 ], with some technical differences, and therefore the two programs should produce similar results. We sought to verify this claim by running the Ahab program (with 1 st and 2 nd order Markov backgrounds), and comparing its performance to that of Stubb. (Ahab can only be run in the local background mode.) Figure 7a shows that there is not a significant difference between Stubb and Ahab CRM predictions. All the above runs were on genomic sequence with tandem repeats masked by the Tandem Repeats Finder program of Benson [ 13 ]. We have found that this heuristic improves genome-wide CRM prediction by Stubb. To substantiate this claim, we ran STUBBSS and STUBBMS on raw (unmasked) genomic sequence. Figure 7b plots the results. We find that both STUBBSS and STUBBMS perform better on masked data than on unmasked data. However, when Stubb is used to analyze shorter sequences (such as the upstream and downstream regions of a gene of interest), we have found unmasked sequence to be more useful, since false positives are less of a concern. Discussion The Stubb program is an extension of Ahab, with the important feature that it can handle two-species data within its probabilistic framework. The two programs differ in their underlying optimization method, with Stubb using an Expectation-Maximization approach in contrast to Ahab's conjugate gradient method. Performance evaluation of the two programs shows little difference between them, implying that the algorithm is robust to the actual optimization method used. Another technical difference between Ahab and Stubb is in the manner that orientation of binding sites is treated. While Stubb assumes a uniform prior on the orientation of a binding site, Ahab picks the best orientation for each site, with the caveat that probabilities are not strictly normalized. An important component of Stubb is the alignment step where the two species are aligned (using LAGAN) and blocks of high sequence similarity are extracted. (See Methods.) The parameters used in LAGAN runs were obtained from Emberly et al . [ 14 ], who derived the alignment parameters that maximize the overlap between experimentally verified binding sites and blocks of sequence conservation. They also studied the effect of changing the alignment algorithm (LAGAN from Brudno et al . [ 7 ]versus SMASH from Zavolan et al . [ 15 ]) for CRM's in the two fly species, and found no significant difference. Finally, the similarity thresholds we use for defining conserved blocks (10 bp or longer, with >70% identity) were obtained by trying a broad range of values, and choosing those that produced the best results, as per our genome-wide evaluation. Tandem repeat masking is a common pre-processing step for many sequence analysis applications involving binding sites. These repeats are short locally duplicated sequences, that may or may not be related to binding sites. It is not clear a priori how tandem repeats should affect module detection – repeats similar to binding sites of the system may improve sensitivity when they occur in CRM's; but if repeats resembling binding sites occur by chance in non-functional regions, prediction specificity may suffer. The occurrence of tandem repeats marks statistical deviation from Stubb's probabilistic model of sequence generation. In our tests, we found that repeats distract the algorithm more than they help, as manifested in better performance on repeat-masked sequence. (See Figure 7b .) This may be because two of the weight matrices in our collection (Hunchback and Caudal) resemble a poly-T stretch. Therefore, the poly-A or poly-T tandem repeats that occur promiscuously in the genome may be confused with sites of these two weight matrices. A recently published tool for genome-wide CRM prediction, called PFR-Searcher (Grad et al . [ 11 ]), first identifies "phylogenetically footprinted regions" or "PFR"s, that are sequences conserved between the two fly species, and then searches for a subset of these that are most similar in content to an input set of promoters. Their approach differs from Stubb in the nature of prior information input to the algorithm. While Stubb uses an input set of weight matrices, the training data for PFR-Searcher is a set of CRM's which, in their approach, is itself provided by a similarity search among PFR's of co-regulated genes. PFR-Searcher therefore has the advantage of not requiring knowledge of the transcription factor weight matrices relevant to the system. However, its ability to predict the binding site composition of potential CRM's is therefore more limited as compared to Stubb. (The Stubb program computes an average "parse" of the predicted module into its constituent binding sites for various transcription factors.) Grad et al . [ 11 ]report an evaluation of their algorithm on a test system very similar to ours, but with enough minor differences to make a direct comparison of performance impossible. For instance, the entire list of CRM's predicted in their evaluation corresponds, as per our CRM → gene mapping, to a set of only 46 unique genes, of which 31 are patterned. Twenty of these 31 correct predictions are also found in the top 46 gene predictions of STUBBMS, indicating a good degree of overlap between the two methods, at least in their highest ranked predictions. A fair and comprehensive comparison of the predictive power of these two algorithms is an interesting topic for future work, and it will be even more interesting to run STUBBMS only on PFR's detected by their criteria. Regarding the recovery of patterned genes by Stubb, several observations can be made. Of the 286 genes with ap patterns, we recover roughly half at a score cut-off of 10, using STUBBMS. Why is the other half not found? While it is obvious that lowering the cut-off will detect more patterned genes, there are other reasons why a patterned gene may be missed by Stubb. Some genes are likely to be lost due to the distance filter we have imposed (CRM to nearest gene <20 kb), since the regulatory regions of some genes (e.g., homeotic genes) are likely to be larger than that. More importantly, most of the patterned genes that are not part of the core transcriptional machinery have derivative patterns that reflect a more complex input (binding sites for pair rule factors, d-v factors etc.) and thus will only be recovered to the extent their input has a solid maternal/gap component. Conversely, there are at least two reasons for reporting false positives (roughly two thirds at a score cutoff of 10). The presence of an insulator could prevent the interaction between a CRM and its nearest basal promoter. More likely is a scenario where the predicted CRM's do drive expression but at post-blastoderm stages. All gap factors are active in multiple tissues in later development and therefore CRM's with dominant or exclusive gap input may well be active in these later contexts. These caveats affect all current CRM detection algorithms, and accounting for such additional axes of information as genomic context and module composition rules will be a difficult but important challenge for the future. A very interesting observation comes from the analysis in Table 1 : Genes predicted by STUBBSS only, and not by STUBBMS, have weak or intermediate expression pattern more often than strong expression. This means that the CRM's that are not well-conserved between the two species (and hence not picked up by STUBBMS) typically correspond to weakly expressed genes. This ties in with previous studies (e.g., Domazet-Loso & Tautz [ 16 ]) that found fast evolving genes in Drosophila to be expressed relatively weakly. The Stubb program not only predicts cis-regulatory modules genome-wide, it additionally outputs the binding site profile of each predicted CRM, i.e., the locations and probabilities of binding sites in the CRM. Schroeder et al [ 5 ] use the corresponding feature in Ahab for a systematic analysis of the composition of all known or validated segmentation CRMs. The use of STUBBMS improves such binding site predictions. It is easy to adapt the program to take as input orthologous CRM's from the two species, and highlight the changes in terms of their binding site compositions. This leads to a powerful bioinformatic tool to predict regulatory changes between the two fly species. We can thus obtain hypotheses about changes in expression patterns, which can be verified experimentally. We have examined a representative collection of CRM's, and experimentally verified several of the changes predicted by Stubb, thereby building a catalogue of the different modes of cis-regulatory evolution. The results of this study will be reported in the near future. Conclusions We have seen that the use of a second fly genome significantly improves genome-wide module prediction. Since STUBBMS uses a natural "two-species" extension of the algorithm of STUBBSS, this finding is largely a statement about the inherent potential of cross-species comparison as a paradigm for improving functional genomics. The STUBBMS program also has a natural extension to incorporate more than two genomes, and it will be very interesting to see how much of a difference a third genome makes. The genome of D. yakuba is expected to be sequenced soon, and since this species is closer to D. melanogaster, it may help better discriminate conserved regulatory modules. Methods Alignment of D. melanogaster and D. pseudobscura D. melanogaster sequences were obtained from Flybase Release 3. The analysis was limited to the five chromosome arms 2L, 2R, 3L, 3R, and X. D. pseudobscura contigs were obtained from (February 2003 Release). Based on Blast results, we created a mapping, called "CONTIGMAP", between regions of the D. melanogaster genome and D. pseudobscura contigs, each region typically being tens of Kb long. This mapping is many to many, i.e., different regions of D. melanogaster may map to the same contig, and the same (or overlapping) region in D. melanogaster may map to two or more D. pseudobscura contigs. For each entry (M, P) in CONTIGMAP, where M is the D. melanogaster region and P is the D. pseudobscura contig, the LAGAN alignment program (Brudno et al . [ 7 ]) was run, with parameters gap start = -6, gap extension = 0, match = 1, and mismatch = -2, and all contiguous ungapped blocks of alignment, with length 10 bp or more and 70% identity or more, were extracted. In cases where the same region in D. melanogaster was mapped to multiple contigs, the density of LAGAN blocks was then used to choose exactly one mapping contig. Stubb runs Tandem repeats in the input sequences were masked with the Tandem Repeat Finder program of Benson [ 13 ], with parameter settings: (match = 2, mismatch = 5, indel = 5, match probability = 0.75, indel probability = 0.2, minimum score = 20, maximum period = 500). STUBBSS was run on the D. melanogaster genome with a sliding window of length 500 bp, in shifts of 50 bp. The input weight matrices for the maternal and gap transcription factors Bcd, Hb, Cad, Kni, Kr, Tll, Dstat and the torRE binding factor were obtained from Rajewsky et al . [ 4 ] and Schroeder et al . [ 5 ]. A weight matrix for the transcription factor Gt was constructed from known functional sites collected from the literature. STUBBMS was run on each entry (M, P) in CONTIGMAP, using a sliding window of length 500 bp on the D. melanogaster sequence M, in shifts of 50 bp. Thus, STUBBMS was not run on regions of D. melanogaster that are not aligned with some D. pseudoobscura contig. The weight matrices used were the same as in STUBBSS runs. The locations of the blocks computed in the alignment step (above) were input to STUBBMS, and the input value of the neutral mutation rate was 0.5, the value being chosen due to its better performance over alternatives tested. Each genome-wide run of Stubb produces, for each starting position of the sliding window, a score that measures the likelihood of the sequence having a cluster of binding sites. The next step is to extract the coordinates of each window that scores better than all other windows overlapping it. Such windows correspond to local "peaks" in the score profile along the genome. All such "peak" windows with scores above a certain threshold are sorted in decreasing order of their score, to produce a sorted list of predicted CRM's. Each window in this list is annotated with useful information including the identity and relative location of its neighboring genes. The list is then filtered to retain only those predicted CRM's where Stubb predicts occurrences of at least two weight matrices. This is a heuristic that incorporates the combinatorial nature of CRM's, i.e., their tendency to have sites for multiple transcription factors (activators as well as repressors.) Finally, any predicted CRM that overlaps with an exon is removed from the list before evaluation. The predictions made by STUBBMS and STUBBSS are listed in the files "Predicted CRM's – two species" ( Additional File 4 ) and "Predicted CRM's – single species" ( Additional File 5 ), respectively. Annotation of gene expression database The 792 genes which the BDGP expression database lists as showing expression during blastoderm (embryonic stages 4–6) were visually inspected. From this list, we removed genes with ubiquitous expression (426; this also removes the presumably very small number of genes whose ubiquitous expression is controlled by separate "regional" modules), extremely faint or irreproducible expression (31), or expression in pole cells or yolk nuclei only (64), as well as genes whose expression is modulated along the dv axis only (13). The remaining 258 genes show patterned expression in the somatic portion along the ap axis of the blastoderm embryo; 28 known segmentation genes not captured in the BDGP expression database were added to the list, for a total of 286 genes showing ap patterned blastoderm expression. These genes were further categorized by expression level (strong, intermediate, weak) and type of pattern (ap, ap+dv, dv+ap). ap includes gap, pair rule and segment polarity-like patterns (e.g., Kr , fkh , eve ); ap+dv denotes ap pattern with some dv modulation (e.g., kni , so , en ); dv+ap denotes dv pattern with some ap modulation (e.g., neur ). Authors' contributions SS and EDS worked out the details of genome-wide Stubb runs. SS performed the Stubb runs, collected all statistics from the runs, and drafted the manuscript. EDS suggested several analyses reported. MDS, UU, and UG annotated the genes for expression pattern, suggested many of the analyses in the Results and Discussion sections, and wrote parts of the manuscript. All authors read and approved the final manuscript. Note added in proof A recently published paper (Berman et al : Genome Biol 2004, 5:R61, published 20 August 2004.) also evaluates the effect of cross-species comparison on CRM prediction in Drosophila. Supplementary Material Additional File 1 Test Genes List of 2167 genes with expression information. (Source BDGP + literature.) Click here for file Additional File 2 Patterned Genes List of 286 genes with blastoderm pattern, as determined by manual inspection of in-situ expression pattern. (Source BDGP + literature.) Click here for file Additional File 3 Additional Genes List of 2065 genes that are ubiquitous, maternal only, or not expressed. (Source BDGP.) Click here for file Additional File 4 Predicted CRM's – two species List of predicted CRM's using two-species Stubb with a global, 2 nd order background, sorted in decreasing order of Stubb score. Click here for file Additional File 5 Predicted CRM's – single species List of predicted CRM's using single-species Stubb with a local, 1 st order background, sorted in decreasing order of Stubb score. This list of predictions, as well as that in Additional File 4 (above), is meant to be as inclusive as possible; therefore, the specificity of the lowest ranked predictions may be poor. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521067.xml
524490	Patterns of Schistosoma haematobium infection, impact of praziquantel treatment and re-infection after treatment in a cohort of schoolchildren from rural KwaZulu-Natal/South Africa	Background Schistosomiasis is one of the major health problems in tropical and sub-tropical countries, with school age children usually being the most affected group. In 1998 the Department of Health of the province of KwaZulu-Natal established a pilot programme for helminth control that aimed at regularly treating primary school children for schistosome and intestinal helminth infections. This article describes the baseline situation and the impact of treatment on S. haematobium infection in a cohort of schoolchildren attending grade 3 in a rural part of the province. Methods Primary schoolchildren from Maputaland in northern KwaZulu-Natal were examined for Schistosoma haematobium infection, treated with praziquantel and re-examined four times over one year after treatment in order to assess the impact of treatment and patterns of infection and re-infection. Results Praziquantel treatment was highly efficacious at three weeks after treatment when judged by egg reduction rate (95.3%) and cure rate of heavy infections (94.1%). The apparent overall cure rate three weeks after treatment (57.9%) was much lower but improved to 80.7% at 41 weeks after treatment. Re-infection with S. haematobium was low and appeared to be limited to the hot and rainy summer. Analysis of only one urine specimen per child considerably underestimated prevalence when compared to the analysis of two specimens, but both approaches provided similar estimates of the proportion of heavy infections and of average infection intensity in the population. Conclusion According to WHO guidelines the high prevalence and intensity of S. haematobium infection necessitate regular treatment of schoolchildren in the area. The seasonal transmission pattern together with the slow pace of re-infection suggest that one treatment per year, applied after the end of summer, is sufficient to keep S. haematobium infection in the area at low levels.	Background Schistosomiasis is one of the major health problems in tropical and sub-tropical countries [ 1 ]. The schistosomiasis endemic area in South Africa is situated in the north-east and covers roughly one quarter of the country, with Schistosoma haematobium being the most common species [ 2 ]. In 1995 it was estimated that more than four million South Africans were infected with schistosomes [ 3 ]. Possible consequences of S. haematobium infection include haematuria, dysuria, nutritional deficiencies, lesions of the bladder, kidney failure, an elevated risk of bladder cancer and – in children – growth retardation. Accordingly the estimates for morbidity and mortality in affected populations are high [ 4 - 7 ]]. School age children usually present with the highest prevalence and intensity of S. haematobium infection [ 8 ]. However, negative health consequences are not limited to this group since high intensity infections can cause serious chronic disease long after initial infection [ 9 ]. Some studies also suggest that schistosomiasis may play a role as a risk factor for HIV infection and that helminth infections in general negatively affect the immune system of HIV infected persons [ 10 , 11 ]. In 1998 the Department of Health of the province of KwaZulu-Natal (KZN) in co-operation with the Department of Education established a pilot programme for helminth control that aimed at regularly treating primary school children for schistosome infections and intestinal helminth infections [ 12 ]. All children in participating schools were treated without prior screening of infection status. The rationale behind this and similar programmes in other countries is not to eliminate infection in a given area, but to keep infection intensities low in this vulnerable age group in order to prevent serious morbidity [ 13 , 14 ]. Our objectives were to describe the pattern of schistosome infection at baseline, to monitor the impact of treatment in our study population and to assess re-infection after treatment in order to develop recommendations for future control activities. Methods Study area, population and treatment The study was conducted in central Ingwavuma district in northern KwaZulu-Natal (Figure 1 ). The study area covers approximately 28 × 16 km on both sides of the perennial Pongola River (Figure 2 ). Climate is tropical to subtropical (Figure 3 ) with a hot and wet summer (November – February) and a cooler and dry winter (June – August). The study population (Table 1 ) was recruited from all ten primary schools in the area. It was limited to children who attended grade 3 at the start of the study in order to keep disturbances of the school routine to a minimum and because this grade should represent the infection situation in a primary school relatively well [ 15 ]. All grade 3 pupils were eligible for participation with one exception: during the baseline survey two out of five and one out of four grade 3 classes in two large schools had to be excluded due to logistic constraints. These classes, however, were included during treatment and successive surveys. All pupils who provided a urine specimen during the pre-treatment survey were included in the analysis of infection patterns at baseline but only children who had been treated with praziquantel were included in the analysis of the post-treatment surveys. Of these, another 4.6% who reported having received additional treatment for schistosome infection while our study was ongoing were excluded from analysis. Otherwise only children who refused to participate or who were absent or unable to produce a specimen during each of our repeated visits were not included in the analysis of the respective surveys. They were included, however, for those surveys where they participated in order not to increase bias due to the likely difference in disease status between absentees and pupils who attended school [ 16 ]. Treatment in all primary schools in the entire district was carried out in April and May 1998 by school nursing teams from the two local hospitals as part of the first treatment campaign of a provincial helminth control programme. The study team assisted the nurses with treatment and also recorded those of the study population who were treated and those who were not. All consenting children from all grades were treated for schistosome infection with a single dose of 40 mg/kg praziquantel (Biltricide ® , Bayer) without regard to infection status. In order to facilitate administration of the drug, the nurses had been provided with a dosing sheet that showed the correct dosage for different bodyweights. The weight of the children was determined using an ordinary bathroom scale. Because one Biltricide ® tablet containing 600 mg of praziquantel can be subdivided into four segments of 150 mg the required dose can be administered relatively accurately. The drug was administered with a glass of water after the children had eaten a peanut butter sandwich which was provided by the treatment team. Children were asked to swallow the tablets with some water in front of one of the team in order to monitor adherence. Because praziquantel is considered a safe drug and has been used extensively since its introduction in the early 80ies possible side effects of treatment were not monitored systematically [ 17 ]. Children were also treated for intestinal helminth infection with 400 mg albendazole (Zentel ® , SmithKline Beecham) and albendazole treatment was repeated in October 1998 [ 18 ]. After the end of the study the participants were included in the normal treatment routine of the control programme. Ethical considerations Ethical clearance was obtained from the Ethics Committee of the Faculty of Medicine of the University of Natal/Durban and the study was also approved by the Central Medical Ethics Committee in Denmark. Before the onset of the study, information meetings were held with the staff and parents of the schools in the study. At these meetings informed consent was obtained from the parents. Informed consent from the children was obtained directly before the first specimen collection. Specimen collection and processing An initial survey in March 1998 to assess S. haematobium infection in the study population was followed by treatment for schistosome and intestinal helminth infections. Follow-up surveys to monitor loss of infection and re-infection were conducted at 3, 16, 41 and 53 weeks after treatment (Table 2 ). Urine specimens were collected between 10:00 and 13:00 hours [ 13 ]. On our visits to the schools, pupils were provided with labelled 500 ml specimen containers and asked to provide a urine specimen. Each school was visited at least three times during each survey in order to include children who were absent or unable to deliver a specimen on the first occasion. Apart from the pre-treatment survey, where only one specimen was collected, an effort was made to obtain two urine specimens from each pupil. However, the results reported in this article are calculated using only the first specimen obtained. Otherwise the lower sensitivity of the pre-treatment survey would have invalidated comparisons with the follow-up surveys. The results of both obtained specimens are used only in Table 2 where we directly compare them to those of only one specimen. Filled specimen containers were brought to the laboratory where filtration of a sub-sample of 10 ml was carried out on the same day [ 19 ]. Specimens of less than 10 ml were measured before filtration and the number of eggs per 10 ml calculated. Before microscopy, eggs were stained using 50% Lugol's iodine saline solution [ 20 ] and then counted by 3 microscopists in order to obtain an indirect measurement of infection intensity. These counts did not differentiate between viable and non-viable eggs. Repeat counts by different microscopists were done on a sub sample of about 5% of the slides for quality control purposes. These counts revealed no bigger discrepancies. Infection intensities are expressed as eggs per centilitre (EPC, 1 centilitre = 10 ml). Statistics Data were double entered, the duplicates compared and corrected for data entry errors. Statistical analysis was carried out in Stata 7 for Windows [ 21 ]. In order to reduce the influence of extreme outliers, geometric means were preferred to arithmetic means to summarise population infection intensity (Table 1 and Table 2 ). Uninfected children were included by adding 1 to all egg counts before log transformation and subtracting it again after re-transformation. However, when comparing intensity, non-parametric statistics were used because even the log transformed egg counts were still far from being normally distributed. Cure rates (CR) and egg reduction rates (ERR) were calculated using the formulae below [ 15 ]: Results Infection patterns at baseline In the pre-treatment survey 68% of the study population were found infected with S. haematobium and 38% (= 56% of the infected children) had egg counts of 50 or more EPC, the WHO [ 4 ] threshold for heavy intensity infections (Figure 4 ). As shown in Table 1 , differences between sexes regarding prevalence and intensity of S. haematobium infection were moderate. Figure 5 demonstrates that age patterns of infection at baseline differed considerably between sexes. S. haematobium prevalence of boys slowly increased from 60% in the youngest to 79% in the oldest age group whereas the youngest girls had a much lower prevalence (37%) but the increase with age was steeper. Treatment Of the 1109 children who participated in the baseline survey only 852 (76.8%) were treated with praziquantel. 228 children (20.6%) did not receive treatment either because they refused to be treated (7 children) or because they were absent on treatment day (221) and for 29 children (2.6%) it is unclear whether or not they were treated. Differences between the treated and untreated groups (excluding children of unclear treatment status) regarding sex, age, infection status and infection intensity were small and not statistically significant. S. haematobium infection status, CR and ERR over 53 weeks after treatment are summarised in Table 2 . Infection intensity measured as the geometric mean EPC had decreased by more than 95% as soon as three weeks after treatment and over the following months a further small decrease is documented until 41 weeks after treatment. The pattern for prevalence and CR of heavy intensity infections is very similar to this. Total prevalence shows the same trend over time, but in contrast exhibits considerably smaller decreases than the other two measures. In Table 2 we also report the results that were obtained when using both specimens that were collected in the post-treatment surveys. When comparing the different approaches it is obvious that – as expected – the use of only one sample considerably underestimates the total prevalence in this population, but that estimates for prevalence of heavy infections and geometric mean intensity are quite similar. Re-infection No discernible re-infection took place between 3 and 41 weeks after treatment (Table 2 and Figure 4 ) whereas the increase between 41 and 53 weeks after treatment was substantial (two-sided p < 0.001 for both prevalence and intensity using the sign test for equality of paired observations [ 22 ]). The group at risk of re-infection (Figure 5 ) was defined as those children who were found uninfected in at least one of the three surveys at 3, 16 or 41 weeks after treatment (n = 796). Fifty-three weeks after treatment 16.8% of these children were found to be re-infected with S. haematobium . The geometric mean EPC including uninfected children of 0.44 was still far below the corresponding figure before treatment (16.08) and this was also true for the geometric mean EPC when excluding uninfected children (58.2 before treatment and 6.6 at 53 weeks after treatment). Figure 5 shows that the age pattern of re-infection in the study population also differed between sexes: it continuously decreased with age for boys but not for girls where re-infection reached a peak in the group that had been 11 to 12 years old at the time of the baseline survey and that was about 12 to 13 years old at the end of the study. Discussion Infection patterns at baseline The results of our baseline survey are in agreement with a survey conducted in the area about 20 years earlier. Schutte et al. [ 23 ] found prevalences of between 55% and 92% in the four surveyed schools that were situated in our study area. The high total prevalence and the large proportion of high intensity infections that we found indicate that according to WHO criteria regular treatment of schoolchildren in the area is indeed necessary [ 4 ]. The different age patterns for prevalence of S. haematobium infection in girls and boys might hint at different water contact patterns. Treatment The proportion of children treated in our study can not be regarded as representative for the control programme in general, because study participants were more informed about it than their schoolmates. However, the fact that only about three quarters of the children were treated is very disappointing. Although only 3 children did not consent to participate in our study, and only 7 of those who consented openly refused to be treated absenteeism was unusually high during the first round of treatment. This improved greatly in the second treatment half a year later, when – according to the opinion of school staff – pupils and their parents had realised that treatment was beneficial and had only mild and transient side effects. Unfortunately this second round of treatment did only include albendazole but not praziquantel because the schedule of the provincial treatment programme only provided one treatment for schistosome infection per year. Praziquantel treatment resulted in drastic reductions of infection intensity and prevalence of heavy infections as soon as three weeks after treatment, which is in accordance with the literature [ 24 ]. The reduction in overall prevalence of less than 60% at three weeks after treatment is however unsatisfactory, even though it improved to about 80% at 41 weeks after treatment. The explanations that the treatment did not work or that its effect was delayed can be excluded because of the high ERR. It seems more likely that the relatively high post-treatment prevalences are not an indication of a high proportion of active infections after treatment, but that they are caused by "old" and mostly non-infective eggs, laid before treatment, that were trapped somewhere in the tissue and are slowly finding their way to the lumen of the bladder [ 25 , 26 ]. Unfortunately our laboratory examination did not differentiate between viable and non-viable eggs, but the data presented in Figure 4 are consistent with the above explanation because very little change is visible between 3 and 41 weeks after treatment with regard to infections of more than 50 EPC. During this period prevalence decreased almost exclusively in the low intensity range. If many active infections (= egg laying schistosomes) had been lost, this should also have had an impact on infections of higher intensity. The high variability of repeated S. haematobium egg counts [ 27 ] renders single egg counts a less than optimal tool for estimating total prevalence and for identifying the infection status of individuals. Our results however show that estimates of the proportion of heavy infections and of population infection intensity are similar to those obtained when examining two specimens. The examination of three or more specimens per child would most certainly have led to even higher estimates of total prevalence but we doubt that it would have changed the other two estimates considerably. All this indicates that the reporting of measures of infection intensity is not only important because they are a better indicator of population morbidity than prevalence [ 8 , 15 ], but that intensity is also a more reliable marker of treatment success defined as the removal of egg-laying worms. This is especially important when relying on single egg counts to assess the effectiveness of the intervention which is usually the case in treatment programmes and larger field studies [ 28 ]. Re-infection Because of the slow decrease in total prevalence after treatment it did not seem appropriate to restrict analysis of re-infection to those children who were egg-negative three weeks after treatment. According to our above reasoning this would have excluded a number of children who had been treated successfully but were still excreting old eggs. On the other hand we did not want to restrict the analysis to those children who were found egg-negative at 41 weeks after treatment. Even though this was the survey where we found the lowest prevalence, we might have excluded children who had been treated successfully, but had become re-infected again before this survey. Therefore we included all children who were found egg-negative at either 3, 16 or 41 weeks after treatment into the group at risk of re-infection. We are, however, aware that this definition is likely to include some uncured children who were still harbouring low level infections. Our data indicate that S. haematobium transmission occurred mainly during the hot and humid summer. According to the literature, S. haematobium has a pre-patent period (infection to egg-excretion by the host) of about eight to ten weeks [ 29 , 30 ]. Thus the surveys at 41 weeks and at 53 weeks after treatment should approximately reflect transmission from early May (treatment) to early December and from early December to late February respectively. The former period covers the South-African, winter, spring and only a short part of the summer whereas the latter covers most of the hottest and wettest part of the year (Figure 3 ), conditions which favour S. haematobium transmission and the development of their Bulinus globosus snail hosts [ 31 , 32 ]. Moreover, children go swimming more frequently because of the hot weather and the long summer holidays in December and January give them ample time to do so. Seasonality of S. haematobium transmission is well documented for the highveld region of Zimbabwe[ 33 ] with patterns rather similar to the ones found here, which seem to be caused by seasonal variation in snail populations as well as human water contact patterns [ 34 ]. A study in southern Natal found that recreational activities accounted for most of the water contact and – unlike household related water contacts – showed strong seasonal variation [ 35 ]. Conclusions Our study shows that according to WHO guidelines [ 4 ] the high prevalence and intensity of S. haematobium infection in the area indeed necessitate regular treatment of schoolchildren, that praziquantel treatment is highly efficacious in reducing the proportion of moderate and heavy infections and that one treatment per year after the end of summer is sufficient to keep infection intensities at low levels. Because levels of S. haematobium infection in the study area seem to be among the highest in the country [ 23 ] the latter would probably also apply to other parts of South Africa. The slow pace of re-infection might suggest that it could even be sufficient to only treat every two years, but this would need to be verified in a separate study that follows treated children over this period. Because we would not want to discourage future attempts to control schistosomiasis in the region and elsewhere we would finally like to stress that the above described money and time consuming intensive examinations are only required when doing research but that for a control programme a minimum of surveillance is sufficient [ 4 ]. Competing interests The authors declare that they have no competing interests. Authors' contributions ES conceived of the study and designed it together with AO, PM, JDK and CCA. ES conducted the field work with contributions from AO, JDK, CCA and WB, did the statistical analysis and drafted the manuscript. All authors contributed to the final version of the manuscript and read and approved it. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524490.xml
449898	Stopping the Rot	Phytophthora species blight potatoes and tomatoes, devastate soybean, rot cacao, and are the cause of sudden oak death. Understanding this versatile genus will be key to its control	In July 2000, the finger of blame for a mysterious mass killer of Californian oak trees came to rest on a previously undescribed plant pathogen. From the initial identification of Phytophthora ramorum , it took less than four years to produce a draft sequence of its genome, one of the fastest-ever discovery-to-sequence stories for a complex pathogen. This achievement was a United States initiative, facilitated by the injection of federal and state funding into Phytophthora research. But the US is not alone in the battle against this genus. Phytophthora species cause thousands of millions of dollars of damage to the world's commercial crops every year: they blight potatoes and tomatoes, devastate the lucrative soybean, and rot cacao, threatening the world's supply of chocolate ( Figure 1 ). But for Sophien Kamoun, Associate Professor of Plant Pathology at Ohio State University (Wooster, Ohio, United States), these destructive organisms present an exciting opportunity. There are some 60 species of Phytophthora , but so little is known about the genus, he says, that there are things about its species we didn't even know we didn't know. ‘What I find really exciting,’ he says, ‘is discovering these unknown unknowns.’ Figure 1 Phytophthora Infestations (A) Potato and (B) cacao pod. (Reproduced courtesy of Sophien Kamoun, Ohio State University [A], and Christopher J. Saunders and the USDA Agricultural Research Service [B].) The most infamous of the Phytophthora pathogens is the potato late blight, P. infestans . It was this species that led to the Irish potato famine in the mid-1840s, which resulted in the death or displacement of millions. Today, P. infestans is estimated to cost potato and tomato farmers US$5,000,000,000 a year in lost revenue. The story for the soybean pathogen P. sojae is similar, causing loss of more than US$1,000,000,000 a year to soybean growers. In addition to the direct economic impact of these pathogens, introduced Phytophthora can cause severe damage to native flora. The most recent Phytophthora on the scene is P. ramorum , which has caused ‘sudden oak death’ (SOD) in tens of thousands of oak trees across the coastal counties of California, is now present in at least three other US states and is threatening to take on the native flora of the entire North American continent. It is also lurking in Europe, although apparently with less devastating consequences. Molecular Machinery With this kind of impact, it's no surprise that money has poured into research on Phytophthora . This year, the US federal government will channel US$7,400,000 toward research into SOD. A major focus of this funding is genomics. Sequencing the genomes of several Phytophthora will help clarify the phylogeny and evolution of these enigmatic organisms ( Box 1 ) and improve methods of detection and identification. Ultimately, however, sequencing should reveal the molecular tricks that this genus uses to subvert the defences of its plant hosts, allowing scientists to come up with new ways to combat these troublesome organisms. The P. infestans sequencing initiative, coordinated by Kamoun, has recently completed a survey sequence of the genome that gives an initial understanding of how this organism is structured. Perhaps most striking is its size. ‘It's a huge genome,’ says Kamoun. At about 250 megabases (Mb), it's about twice the size of the Arabidopsis genome. His latest research, published in the Journal of Biological Chemistry ( Tian et al. 2004 ), describes a P. infestans protease inhibitor—extracellular protease inhibitor 1 (EPI1)—that could be one of a unique class of suppressor proteins that Phytophthora deploy to infect and counteract host defences. The pathogen seems to upregulate the epi1 gene during colonisation of its host. EPI1 inhibits plant apoplastic proteases—extracellular enzymes that are part of the host's defensive armoury that have evolved to prevent foreign proteins entering cells. ‘Based on its biological activity and expression pattern, EPI1 may function as a disease effector molecule and may play an important role in P. infestans colonisation of host apoplast,’ Kamoun and his colleagues report ( Tian et al. 2004 ). If further research confirms this function for EPI1, then it will become one of just a handful of pathogen molecules that have been shown to suppress host plant defenses. A search in sequence databases for matching motifs reveals just one similar sequence in the entire bacterial and fungal kingdoms. However, apicomplexans like Toxoplasma gondii that transit through the mammalian digestive tract also appear to secrete protease inhibitors allied to EPI1. This similarity suggests an analogy between plant apoplasts and mammalian digestive tracts. Both environments are rich in proteases, but nevertheless are colonised by a variety of microbial pathogens. In the case of an apoplast, the pathogen is P. infestans , whilst in the mammalian gut, it's T. gondii —and although they are phylogenetically distant, these pathogens seem to have independently recruited similar secreted proteins to inhibit the defensive proteases produced by their hosts. Interestingly, whilst T. gondii inhibitors inhibit gut enzymes trypsin and chymotrypsin, EPI1 does not, suggesting that coevolution between the inhibitors and their target proteases may shape the specificity of these pathogenic enzymes. Armed with this new insight into the molecular cunning of P. infestans , Kamoun hopes that it will be possible to come up with ways of slowing disease progression. Importantly, it looks likely that protease inhibitors such as EPI1 are present in other Phytophthora species. There are significant matches between the epi1 gene sequence and motifs from at least five other closely related species. So any methods of blocking the action of protease inhibitors in P. infestans might also work against other Phytophthora . The protease inhibitors are one of Kamoun's ‘unknown unknowns’. ‘It's an example of something that we had absolutely no idea was in the genome,’ he says. Even more advanced than the P. infestans genome project is an ongoing collaboration between the Virginia Bioinformatics Institute (Blacksburg, Virginia, United States) and the US Department of Energy's Joint Genome Project based in Walnut Creek, California. The focus here is the soybean pathogen P. sojae and the SOD pathogen P. ramorum , for which draft sequences are now complete ( www.jgi.doe.gov/ ). The genomes are much smaller than that of the 250-Mb P. infestans — P. sojae is about 90 Mb and P. ramorum is just 55 Mb. This, in part, explains the comparative speed of these sequencing efforts. But another factor is undeniably the fear of the unknown P. ramorum , which in 2002 netted the Virginia–California initiative US$3,800,000 in federal funding to describe its genome. Ultimately, however, the sequence of one species will help to inform on the sequence of other related species. Brett Tyler, Research Professor at the Virginia Bioinformatics Institute, is focusing on the molecular tools used by P. sojae to infect its host. He agrees with Kamoun that understanding this machinery is the way to devise new control measures that could give plants the upper hand in the evolutionary arms race against their Phytophthora pests. At present, most strategies to limit the damage caused by species like P. infestans and P. sojae rely on developing resistant cultivars by selective breeding of varieties with major resistance genes—single genes that can block a pathogen. However, Phytophthora seem able to find ways to overcome these efforts. ‘ P. infestans is absolutely notorious for its ability to genetically change in response to a major resistance gene,’ says Tyler. ‘Typically major resistance genes in potato barely last a single season.’ Things look better for the soybean. New cultivars containing major resistance genes show resilience to P. sojae for five to ten years. However, this resistance is starting to break down, and breeders are running out of major resistance genes with which to conjure new varieties. The alternative, says Tyler, is quantitative or multigenic resistance, which relies on getting plants to express several resistance genes at once, each of which makes a small contribution to the plant's overall resistance. It should be much harder for P. sojae to evolve a new attack against this kind of robust defence. The search is also on for new genes that could be used to encourage quantitative resistance to host species. One particularly promising approach is to pit pathogen against host to see which genes are switched on. The host should upregulate genes that defend it against infection and the pathogen should upregulate genes that it needs to attack. The discovery that plants produce proteases and that Phytophthora have responded by secreting protease inhibitors to disable them is important if long-lasting solutions are to be found. ‘We just need to find a way to introduce new protein-degrading enzymes into the plant that the pathogen doesn't know how to block,’ says Tyler. ‘With these genomic tools we can really accelerate the pace at which we can evaluate different possible protective measures.’ Epidemiology: Identifying the Culprit An additional benefit of the abundance of genetic information is that species identification is becoming increasingly sophisticated. At a glance, Phytophthora can be mistaken for a fungus, so DNA profiling of isolates is crucial if species and strains of species are to be identified correctly so that action appropriate to each infection can be taken. Since 2000, Matteo Garbelotto, Adjunct Professor of Mycology and Forest Pathology at the University of California at Berkeley (Berkeley, California, United States), has spent a significant part of his working life tracking the spread of P. ramorum , the Phytophthora that has killed off vast tracts of oak trees in native Californian forest ( Box 2 ). DNA analysis has been crucial to confirm suspected cases of P. ramorum , and has now revealed that infections have reached at least three other US states. This spread is probably due to the movement of infected ornamentals like rhododendron ( Rhododendron spp.) and viburnum ( Viburnum spp.), which seem to act as carriers for the pathogen. ‘What we're seeing is a parallel to what has been happening throughout Europe, where the infection has basically moved using the commercial routes of the ornamental plant industry,’ Garbelotto says. P. ramorum in Europe P. ramorum does not appear to have the same devastating consequences in Europe as it does in the US—at least not yet. It's not entirely clear why, but it could have something to do with the structure of the bark of different host species, suggests Garbelotto. ‘We normally see more infection where we have more corrugation, and that's because water accumulates in the fissures … where the zoospores have a chance to infect the bark.’ However, he notes, European beech ( Fagus sylvatica ) appears to be extremely susceptible to P. ramorum . ‘If it reaches areas where there are a lot of beeches, it could potentially mirror what's happening in California,’ he warns. In Europe, symptoms characteristic of P. ramorum infection were first described on rhododendrons in The Netherlands in 1993. Once this was confirmed to be the same species as the pathogen responsible for Californian SOD, there was speculation that P. ramorum had either been introduced to the US from Europe or vice versa. However, in December 2002, it emerged that these two populations are of different mating types—A1 in Europe and A2 in the US. The latest research from Garbelotto and his colleagues, due to be published in Mycological Research ( Ivors et al. 2004 ), supports this interpretation, demonstrating that although they belong to the same species, A1 and A2 are distinct lineages and have not exchanged genes for a long time. But last year, a batch of isolates from infected camellias ( Camellia spp.) and rhododendrons in a nursery in Washington state showed that the A1 and A2 strains were living side by side. ‘That was a big surprise for us,’ recalls Garbelotto. ‘We had no knowledge at that point that both the European and the North American type could be present in the same nursery.’ The Threat of Recombination Hybridisation between different species of Phytophthora can produce a new species with different properties from those of either parent. One of the best cases comes from Europe, where a new Phytophthora emerged in 1993 that began to attack alder trees ( Alnus spp.). Research carried out by scientists in the United Kingdom demonstrated that the alder Phytophthora was a product of a hybridisation event between P. cambivora and an unknown species similar to P. fragariae , neither of which attacks alder. Given this propensity for Phytophthora species to hybridise and new phenotypes to emerge, there is legitimate concern that sexual recombination between the A1 and A2 mating types could produce something more devastating than either form. Within the controlled confines of his laboratory, Garbelotto has been exploring whether the two types of P. ramorum can get it together. Initial findings are that oospores—the product of sexual recombination—are being produced, although most of them abort before they reach maturity. However, 30% progress further, and (microscopically, at least) look like they could be functional. ‘They'll germinate,’ he predicts. The threat that hybridisation could create a novel strain with a different host range is a concern that the UK is also taking seriously. In December 2003, an entirely new Phytophthora was isolated from two sites in England. Although the new species—currently referred to as Phytophthora taxon C sp. nov. ( P . taxon C)— appears to cause relatively mild damage to its beech and rhododendron hosts, the UK's Department for Environment, Food and Rural Affairs (DEFRA) acknowledges that hybridisation of P . taxon C with P. ramorum could have serious consequences. ‘The potential for the pathogen to adapt further to its putative new environment intrinsically or via hybridisation is not known,’ note the authors of a DEFRA report on the mystery species ( www.defra.gov.uk/planth/pra/forest.pdf ). ‘Long-distance spread could easily occur through the movement of infected stock of rhododendron or beech and possibly other (as yet unknown) hosts,’ they warn. DEFRA is monitoring the situation closely ( Figure 4 ). However, on the basis of a preliminary DNA analysis, P . taxon C and P. ramorum are only distantly related, making hybridisation unlikely, says Joan Webber, Head of Pathology at the UK government's Forestry Commission. The closest known relative of P . taxon C is P. boehmeriae , a pathogen that has been recorded on several species of tree in China and Australia, and on cotton in China and Greece, suggesting possible origins for the newly described species. But, says Webber, the sequence match between P . taxon C and P. boehmeriae is only 92%—not especially close. Much more evidence is needed to build a strong case for the origin of this new Phytophthora , she says. Figure 4 Infection of P. ramorum at a site in the UK DEFRA is monitoring closely for signs of hybridisation with P . taxon C sp. nov. Origins Indeed, it has taken more than 150 years to track down the geographical origin of the P. infestans strain that caused the Irish potato famine. Jean Beagle Ristaino of North Carolina State University (Raleigh, North Carolina, United States) is due to publish in Mycological Research an analysis of DNA extracted from diseased potato plants preserved from the nineteenth-century Irish epidemic ( May and Ristaino 2004 ). It had long been suspected that the famine was caused by the Ib strain of P. infestans , which is presumed to have originated in Mexico. However, Ristaino's molecular evidence spotlights the Ia strain and traces its probable roots to the Andes. The infection could have found its way from South America to Europe and the US via exports of potato seed on steamships, she speculates. This kind of forensic treatment is more than just interesting. Tracing a Phytophthora species to its site of origin could reveal what keeps them at bay in the areas where they are native, and might suggest new ways to manage them when they are introduced to a different ecosystem, says Garbelotto: ‘There's a huge amount of information that can be learned from understanding where they're coming from.’ So where do pathogens like P. ramorum originate? The best lead, Garbelotto says, is the ease with which it infects rhododendron. These ornamentals are natives of Asia, but there are only a few places where the climate would suit P. ramorum . The most promising, he suggests, are the Southern Himalayas, the Tibetan plateau, or Yunnan province in China. But these are big places, and Garbelotto has plenty on his plate in his battle against the Californian SOD. ‘I am not very hopeful that we'll ever be able to find out where it comes from,’ he says. Figure 2 Reproductive Structures of the Phytophthora The asexual (A) sporangia, (B) zoospores, and (C) chlamydospores, and the sexual (D) oospores. (Reproduced courtesy of Matteo Garbelotto, UC Berkeley [A, D], and Edwin R. Florance, Lewis & Clark College [Portland, Oregon, United States] and the USDA Forest Service Pacific Southwest Research Station in Albany, California [B, C].) Figure 3 Coast Live Oaks Plagued by P. ramorum , Marin County, California (Reproduced courtesy of Matteo Garbelotto, UC Berkeley.) Box 1. A Closer Look at Phytophthora Phytophthora belong to the Kingdom Stramenophiles, so are most closely related to brown algae and diatoms. Their hyphal growth and variety of spores are morphologically and physiologically similar to fungi, for which they are occasionally mistaken, but their parasitic lifestyles have independent evolutionary origins, and therefore they have alternative mechanisms of pathogenicity. Within the class Oomycetes, which comprises all manner of heterotrophic blights, mildews, and molds, the Phytophthora —from the Greek for ‘plant destroyer’—is a pernicious genus, costing the world's farmers thousands of millions of dollars each year in control measures and lost yield. The majority of the 60 or so Phytophthora species that have been described are distinguished by their complex life histories, with both asexual and sexual phases and a bewildering array of associated reproductive structures. Sporangia are asexual spores that provide the pathogen with a short-lived mode of transmission. They are either broken off from the filamentous hyphae to become airborne as in the potato blight P. infestans ( Figure 2 A) or remain attached and divide into swimming zoospores following rains ( Figure 2 B). By contrast, chlamydospores are asexual structures that are adapted for longterm survival ( Figure 2 C). In the case of the Phytophthora causing ‘sudden oak death’ ( P. ramorum ) and forest dieback ( P. cinnamomi ), the chlamydospores play a crucial role, allowing the pathogen to survive from season to season. The formation of sexual structures is relatively rare, but oospores—the product of sexual recombination—occur in many species ( Figure 2 D). Some, such as P. sojae , are known as homothallic, with a single form that has both male and female reproductive structures, whilst others like P. ramorum are heterothallic and require two different mating types to meet for sexual recombination to occur. Box 2. Sudden Oak Death in California and Beyond In 1995, trees in oak forests in the coastal counties of California started to show a range of alarming and lethal symptoms. Since then, the disease has reached epidemic proportions, with tens of thousands of trees dying along approximately 300 km of the central Californian coastline ( Figure 3 ). By 2000, the pathogen responsible had been identified as P. ramorum , and it is now clear that this pest has an extremely broad host range that extends to almost all woody plant species in the coastal forests of California. Many oak hosts suffer lethal branch or stem infections, whilst non-oak hosts only carry mild leaf or twig infections. The species hardest hit include tanoak ( Lithocarpus densiflora ) and the true oaks coast live oak ( Quercus agrifolia ), California black oak ( Quercus kellogii ) and Shreve's oak ( Quercus parvula var. shrevei ). These species develop large wounds or cankers in their woody tissue, which disrupt physiology and in extreme cases lead to death in a matter of months. In non-oak species, P. ramorum does not appear to cause the same damage, leaving hosts like the Californian bay laurel ( Umbellularia californica ) and rhododendrons ( Rhododendron spp.) with the relatively mild symptoms of leaf blight and occasional branch dieback. However, these non-oak hosts probably act as reservoirs of disease and may even help it to spread. The sporangia and chlamydospores that are thought to be the main asexual propagules of the pathogen ( Box 1 ) are readily produced on the foliage of such non-oak species, but do not seem to appear on the bark of most of the infected oak hosts. In March 2004, P. ramorum was found at a large wholesale horticultural nursery in Los Angeles County. The California Department of Food and Agriculture, the US Department of Agriculture's Animal and Plant Health Inspection Service, and state agriculture departments around the country are tracing all plants shipped from this nursery over the past year in an effort to identify and destroy any infected material, and hence prevent any further spread of the disease. However, the rot has already set in at nurseries in three states outside California: Oregon, Washington, and Florida. A National SOD Nursery Survey is underway to try to assess how far the disease might have spread.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC449898.xml
517728	Polyphosphoinositides suppress the adhesion of Haemophilus influenzae to pharyngeal cells	Background One of the primary causes of otitis media (OM), an inflammation of the middle ear, is the bacterium Haemophilus influenzae (HI). OM often occurs to young children, and is mostly treated with antibiotics. Due to concerns over bacterial resistance toward antibiotics, reliable prophylactic treatments such as administrating anti-adhesion agents are now viewed as viable alternatives. Results The present study tested the feasibilty of using phosphoinositides as anti-adhesion agents against HI cells. Cells of non-typeable HI were radiolabeled with 111- indium-oxine, pre-incubated with various individual phosphoinositides for 15 minutes at 37°C, and incubated with a monolayer of human pharynx carcinoma (DT 562) cells for 20 minutes at 37°C. The result showed that at 0.1 mg/mL dipalmitoylphosphatidylinositol-3,4-diphosphate (PI-3,4-PP) had the highest anti-adhesion activity, followed by phosphatidylinositol-3-phosphate (PI-3-P) and phosphatidylinositol-4-phosphate (PI-4-P). The anti-adhesion activity of PI-3,4-PP was dose-dependent ranging from 0.006 to 0.1 mg/mL. In addition, results from an in vivo study demonstrated that pre-incubation of HI cells with PI-3,4-PP at 1 mg/mL suppressed the growth of HI in nasopharynx of neonatal rats. Conclusions These findings suggest that PI-3-P and PI-4-P and more so PI-3,4-PP may serve as prophylactic agents against HI adhesion and colonization.	Background Otitis media (OM) is an inflammation of the middle ear, often seen in children younger than six year of age. OM is caused by infection of nasopharyngeal cells by the bacterium Haemophilus influenzae (HI). Complications of OM include permanent hearing loss and perforation of the tympanic membrane. Generally, OM is treated with antibiotics such as penicillin derivatives. However, in spite of the effectiveness of antibiotic prophylaxis, the increasing bacterial resistance to antibiotics has caused some concerns. This has prompted the development of anti-adhesive agents against HI infection [ 1 ]. Today, several anti-adhesive agents such as xylitol and oligosaccharides have been studied in clinical trials [ 2 - 5 ]. Human casein has been shown to have an inhibitory effect on the adhesion of HI to human respiratory tract epithelial cells [ 6 ], but the active factor(s) has not been characterized. Recently, we have discovered that certain rice flour extract inhibits HI adhesion [ 7 ]. Results from the preliminary purification process indicated that the active factor(s) in the rice flour extract was amphiphilic and structurally resemble to the phosphoinositides. To verify this view, we examined in this study the anti-adhesion activities of phosphoinositides against HI using the models of human pharynx carcinoma (DT 562) cells and neonatal rats. Results and Discussion Bioactivity of polyphosphoinositides The attachment (adhesion) of bacteria to a mammal's nasopharynx area is believed to be the first stage of the bacterial infection, which can lead to OM and other disorders and diseases caused by HI. For this study, in vitro effect of various phosphoinositides on the attachment of HI to nasopharyx was evaluated. The activity was defined as the percent of inhibition of HI adhesion to human pharynx carcinoma cells as compared to the control. Results in Table 1 show that four phosphoinositides, i.e., Ins-1,2,6-PPP, Ins-1,3,4-PPP, GPI and PI at a concentration of 0.1 mg/mL exerted no effect on HI adhesion to the human pharynx cell cultures. On the other hand, PI-3-P and PI-4-P showed 24% inhibition of HI adhesion. More, PI-3,4-PP at the same concentration showed 74% inhibition against HI adhesion. A dose-dependent inhibition of HI adhesion by PI-3,4-PP was observed between 0.006 and 0.1 mg/mL, which exerted 31 to 81% inhibition (Figure 1 ). Table 1 Bioactivities (% inhibition) of phosphoinositides against the adhesion by Haemophilus influenzae Compound % Inhibition Dose (mg/mL) Ins-1,2,6-PPP 6 a 0.1 Ins-1,3,4-PPP -1 0.1 GPI -3 0.5 PI -2 0.1 Pl-3-P 24 0.1 Pl-4-P 24 0.1 PI-3,4-PP 74 0.1 a Within statistical error; thus no activity is presumed. Figure 1 The dose response curve of PI-3,4-PP against the adhesion by H. Influenzae . In Vivo Activity of PI-3,4-PP The inhibitory activity of PI-3,4-PP against the attachment of nontypeable HI was further demonstrated in two trials using a neonatal rat model. Figure 2 shows the average inoculum dose (cfu) and the average number (log 10 [cfu/mL]) of HI recovered for a total of 10 rat pups for each treatment. In trial 1, at the inoculum dose of approximately 100 cfu/pup, the treated group (PI-3,4-PP) containing 1 mg/mL of PI-3,4-PP showed an 80-fold (1.9 logs) reduction in the number of bacteria recovered 24 hours post-inoculation as compared to the control group (HBSS). In trial 2, the rat pups were exposed to much higher doses (approximately 700 cfu/pup) of bacteria. The treated group (PI-3,4-PP), which was protected by the same level of PI-3,4-PP, showed a 4-fold (0.6 log) reduction in the number of bacteria recovered. The result demonstrates that PI-3,4-PP can inhibit the attachment and thus the growth of nontypeable HI in the nasopharynx of neonatal rats. Figure 2 In vivo activity of 1 mg/mL of PI-3,4-PP against the adhesion by H. influenzae . The exact mechanism for the anti-adhesion activity of the phosphoinositides is not known at this time. However, the anti-adhesion activity may be associated with the structures of the phosphoinositides. Molecules containing no phosphatidyl group such as Ins-1,2,6-P, Ins-1,3,4-P and GPI, exerted no effects on adhesion. PI, which contains a phosphatidyl group, also had no activity. In addition to the presence of a phosphatidyl group, phosphorylation of inositol seems to be required for the bioactivity. Since PI-3,4-PP had a higher anti-adhesion activity than PI-3-P and PI-4-P, it is possible that the presence of extra phosphate groups at the positions 3 and 4 of the inositol moiety may exert an even greater effect. Results from a preliminary study (Jeffrey Baxter, personal communication) showed the presence of multi-phosphate groups in a molecule (such as phytate, inositol phosphate, and phosvitin) were inhibitory to HI adhesion. On the other hand, results from our study showed that PI-3,4-PP was more active than phytate (a myo -inositol hexaphosphate), myo -inositol penta-and tetra-phosphates (data not shown). Taken together, these results suggests that the presence of both a phosphatidyl group and an additional phosphate group is essential for the high anti-adhesion activity of PI-3,4-PP as compared to PI-3-P and PI-4-P. It is known that bacterial adhesion involves specific recognition of carbohydrate receptors by pathogen proteins [ 4 ]. This specificity is probably one of the main factors that dictate in which tissue that pathogen species can successfully colonize. Previously, the anti-adhesion effect of some oligosaccahrides has been attributed to their bindings to the specific binding sites of pathogen's proteins [ 4 ]. These oligosaccharides serve as decoys and occupy bacteria's carbohydrate-binding proteins, and thus reduce the binding of pathogens to the native carbohydrate in epithelial cell membrane. Similarly, polyphosphoinositides present in inner ear tissue and kidney [ 8 , 9 ] and other tissues have been postulated as in vivo receptor for aminoglycoside antibiotics [ 10 ]. It is possible that phosphorylated phosphatidylinositol such as PI-3-P, PI-4-P, and PI-3,4-PP may serve as decoys by occupying the binding sites in bacteria and prevent their attachment to the epithelial cells. It has also been reported that phosphatidylinositides can bind to mCD14, a cell-surface receptor on the membrane of monocytes and neutrophils [ 11 ]. Among those tested phosphatidylinositides, PI-3-P, PI-4-P, PI-3,4-PP, and PI-4,5-PP display the highest affinities for mCD14. Recently, non-typeable HI have been shown to adhere to human bronchial epithelial cells through the lipooligosaccharide (LOS) on the cell surface of the bacteria [ 12 ], and LOS further interacts with the platelet-activating factor (PAF) receptor to initiate host cell signal cascade and bacterial invasion [ 13 ]. It is then possible that phosphatidylinositides occupy the binding sites of the bronchial epithelial cells through binding to the membrane of these cells, thus reducing the number of binding sites available for HI attachment. By suppressing the binding of HI to the cell membrane, phosphatidylinositides prevents the replication of the bacteria. Conclusions In conclusion, the PI-3,4-PP suppressed the adhesion of nontypeable HI to nasopharyngeal cells of neonatal rats, and thus preventing the replication of the bacteria in the animal. The results suggest that the phosphoinositides may be used to formulate pharmaceutical and nutritional compositions for prophylactic treatments of OM and other infections caused by HI. Materials and Methods Phosphoinositides Five different classes (I through V) of phosphoinositides were used for this study (Figure 3 ). Class I included 1-D- myo -inositol-1,2,6-triphosphate sodium salt (Ins-1,2,6-PPP) and 1-D- myo -inositol-1,3,4-triphosphate sodium salt (Ins-1,3,4-PPP) (Figure 3A ). Class II included 1-(α-glycerophosphoryl)-D- myo -inositol lithium salt (GPI), class III L-α-phosphatidylinositol ammonium salt (PI), class IV dipalmitoylphosphatidyl-inositol-3-phosphate ammonium salt (PI-3-P), and L-α-phosphatidylinositol-4-monophosphate sodium salt (PI-4-P), and class V dipalmitoylphosphatidylinositol-3,4-diphosphate ammonium salt (PI-3,4-PP) (Figure 3B ). Ins-1,2,6-PPP, Ins-1,3,4-PPP, PI-3-P, and PI-3,4-PP were obtained from Matreya Inc. (Pleasant Gap, PA), GPI from Calbiochem-Novabiochem Corp. (La Jolla, CA), and PI and PI-4-P from Sigma Chemical Co. (St. Louis, MO). All chemicals are reagent grade with purity greater than 99%. Figure 3 Structural formulas of the five (I through V) classes of phosphoinositides. Cell Cultures The Detroit 562 human pharynx carcinoma cell line (DT 562) was obtained from the American Culture Type Collection. The DT 562 cells were seeded into Costar 96-well plates (Corning Life Science, Acton, MA) at a density of 20,000 to 25,000 cells per well, and cultured in Dulbeco's modified Eagle Medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT). The plates were incubated in a humidified atmosphere of 95% air: 5% CO 2 at 37°C until reaching at least 90% confluency. Plates were washed three times with 20 mL of Hanks Balanced Saline Solution (HBSS) (Sigma Chemical Company, St. Louis, MO) to remove serum proteins. Radiolabeling of HI bacteria For the adhesion study, an HI nontypeable bacterial strain was used. The HI isolated from the middle ear of an infected child was a gift from Dr. Lauren Bakaletz of The Ohio State University, Columbus, Ohio. HI was streaked onto Chocolate agar plates (Becto Dickinson Diagnostic Instrument System, Sparks, MD) from frozen aliquots of a low passage number. The plates were then incubated at 37°C in a humidified atmosphere of 95% air: 5% CO 2 for 18 hours. Bacteria were then harvested in phosphate buffered solution (PBS) supplemented with 0.05% bovine serum albumin (BSA) (Miles Inc., Kankakee, Ill.). After centrifugation, the cell pellets were resuspended in a volume of PBS/BSA yielding an optical density of 2.4 at a wavelength of 660 nm. The bacteria were then radiolabelled with 111- Indium-oxine ( 111- In), a high energy, short-lived tracer. Fifty μCi of 111- In solution was added to 2.5 mL of the bacterial suspension and incubated for 20 minutes at 37°C. The radiolabeled bacteria were then washed two times with 10 mL HBSS and unbound 111- In were removed by centrifugation. The bacteria pellets were then resuspended in 5 mL HBSS supplemented with 30 mM 2-hydroxyethyl-piperazine-N'-2-ethane sulfonic acid buffer (Life Technologies, Calsbad, CA). Adhesion Quantitation Prior to adhesion test, aliquots (25 μL) of the 111- In-labeled bacterial suspension were pre-incubated with 25 μL of the test chemical (containing various phosphoinositides) in a Costar polypropylene 96-well plate for 15 minutes at 37°C to allow binding of the test agent to the HI. For adhesion quantitation, aliquots (25 μL) of the pre-incubated mixture were pipetted into the wells of an assay plate containing the DT 562 human pharynx carcinoma cells. The assay plate was incubated for about 15 to 20 minutes at 37°C to allow adhesion of the bacteria to the cell monolayer. Nonadherent bacteria were removed by washing the plate three times with HBSS. The cell monolayer and the adhering of the HI cells were disrupted by the addition of 100 μL of 0.05 N sodium hydroxide. The contents of each well were placed in Cobra polypropylene tubes and the radioactivity counted on a Cobra Gamma Counter (Packard Instrument Co., Meriden, CT). After calibration of the background, the average radiation count of four replicates (per sample) was calculated. The percents of inhibition of bacterial adhesion, as compared to bacterial attachment in control wells containing no test chemical were then calculated. In Vivo Activity A neonatal rat model [ 14 , 15 ] was modified for testing the in vivo activity of PI-3,4-PP against nontypeable HI. Prior to the test, overnight cultures of nontypeable HI were prepared, washed twice and diluted with HBSS to obtain a bacterial suspension of less than 10 5 colony-forming-units (cfu) per mL. Three different test chemicals were prepared for this test: (i) HI + PI-3,4-PP, an aliquot (0.5 mL) of PI-3,4-PP (2 mg/mL in HBSS) mixed with 0.5 mL of the diluted bacterial suspension (5 × 10 4 cfu/mL) and incubated for one hour at 37°C; (ii) HI + HBSS, This bacterial control prepared by mixing and incubating 0.5 mL HBSS with 0.5 mL of the diluted bacterial suspension; and (iii) HBSS, the solvent blank prepared by incubating 1 mL of HBSS under the same conditions. A 10 μL of the test material was used to inoculate 24-hour-old or younger Sprague Dawley rats (Charles River, Portage, MI) by intranasal administration. Twenty-four hours after the inoculation, samples of nasopharyngeal fluid were collected by the slow instillation of 25 μL of HBSS into the left naris, and the initial 10-μL discharge from the right naris was collected for plate count. This procedure insured that the fluid had passed through the nasopharynx. The nasal wash was then spread (or diluted and then spread), onto Chocolate agar plates. The plates were incubated at 37°C overnight and counted for the number of cfu's, an indicator of the number of viable bacteria. Authors' contributions YSH conceived of the study. JWL screened and identified the phosphoinositides, and drafted the manuscript. SNA carried out cell adhesion assay. JM carried out assay for in vivo activity. SMH and PM participated in experiment coordination.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC517728.xml
546215	Quality of care for hypertension in the United States	Background Despite heavy recent emphasis on blood pressure (BP) control, many patients fail to meet widely accepted goals. While access and adherence to therapy certainly play a role, another potential explanation is poor quality of essential care processes (QC). Yet little is known about the relationship between QC and BP control. Methods We assessed QC in 12 U.S. communities by reviewing the medical records of a randomly selected group of patients for the two years preceding our study. We included patients with either a diagnosis of hypertension or two visits with BPs of ≥140/90 in their medical records. We used 28 process indicators based on explicit evidence to assess QC. The indicators covered a broad spectrum of care and were developed through a modified Delphi method. We considered patients who received all indicated care to have optimal QC. We defined control of hypertension as BP < 140/90 in the most recent reading. Results Of 1,953 hypertensive patients, only 57% received optimal care and 42% had controlled hypertension. Patients who had received optimal care were more likely to have their BP under control at the end of the study (45% vs. 35%, p = .0006). Patients were more likely to receive optimal care if they were over age 50 (76% vs. 63%, p < .0001), had diabetes (77% vs. 71%, p = .0038), coronary artery disease (87% vs. 69%, p < .0001), or hyperlipidemia (80% vs. 68%, p < .0001), and did not smoke (73% vs. 66%, p = .0005). Conclusions Higher QC for hypertensive patients is associated with better BP control. Younger patients without cardiac risk factors are at greatest risk for poor care. Quality measurement systems like the one presented in this study can guide future quality improvement efforts.	Background Hypertension affects approximately 58 million Americans [ 1 ]. Lowering diastolic blood pressure (BP) by 10 mm Hg can reduce the number of strokes by as much as 56% and the incidence of coronary heart disease by 37% [ 2 ]. In addition, it has been shown that lowering systolic BP to 150 mm Hg decreases the incidence of all types of strokes [ 3 ]. Although treatment reduces mortality, morbidity and costs, nearly half of all people with hypertension go untreated and only 23% control their BP to the recommended level [ 4 ]. The high prevalence of uncontrolled hypertension is due in part to a lack of awareness: 32% of people with the disease do not know they have it [ 4 ]. However, over 40% of diagnosed hypertensive patients remain uncontrolled [ 4 ]. One potential explanation for uncontrolled hypertension is suboptimal quality of care. Although the U.S. Joint National Committee on Prevention, Detection, and Treatment of High Blood Pressure (JNC-VII) [ 5 ] has codified standards for clinical processes in hypertension in the United States, studies dating back to the 1970s have shown that many patients fail to receive this essential care [ 6 - 8 ]. On the other hand, many of these studies have not established a relationship between these care processes and BP control. One exception is a recent study of U.S. Veterans Administration patients in five facilities, which found a correlation between aggressive treatment and better-controlled BP [ 9 ]. Still, most studies examining the link between care processes and controlled hypertension generally have been confined to single delivery systems, a limited number of facilities, or a relatively small set of indicators of hypertensive quality. In a previous study, we examined general measures of hypertensive quality (including treatment, diagnosis, and follow-up indicators) and found that these care processes were associated with BP control in young women participating in a single health plan [ 10 ]. Studies with more generalizable target populations are lacking. If deficits in process quality are indeed related to BP control, then which patients are failing to receive optimal care? The literature suggests that ethnic minorities and older patients are less likely to have controlled hypertension [ 4 ], but we do not know what clinical factors may be affecting patient care. For instance, physicians may be targeting higher-risk patients and administering better care to those with diabetes, coronary artery disease (CAD), and tobacco abuse. Likewise, providers may be delivering better care to older patients as they are also at higher risk, though limited evidence suggests the opposite is true [ 11 , 12 ]. We hypothesized that overall process quality for hypertensive care is associated with better BP control. We developed indicators of hypertensive care and determined whether patients had received the indicated care by reviewing medical records for a national sample of patients receiving care in a variety of settings. We also investigated whether patients with other cardiac risk factors received better hypertensive care and had better BP control. Methods Design and sampling Our examination of hypertensive care was part of a larger study called the Community Quality Index Study (CQI), a cross-sectional study that assessed effectiveness of care for 32 different clinical conditions by examining medical records for patients in 12 randomly selected communities with populations greater than 200,000 (Boston, Cleveland, Greenville, Indianapolis, Lansing, Little Rock, Miami, Newark, Orange County, Phoenix, Seattle, and Syracuse) [ 13 ]. The methodology and overall results from CQI are presented elsewhere, and are summarized briefly here [ 14 ]. Between October 1998 and August 2000, study participants were selected by random digit dialing and asked permission to obtain copies of their medical records from all providers they had seen in the previous two years [ 14 ]. Of the 20,158 persons in the starting sample, we excluded 2,091 (10%) because they had moved out of the area, passed away, or become incapacitated in some manner that left them unable to participate in the study. Of the 17,937 adults who were eligible for the study, 74% (13,275) completed the telephone survey. Of the 12,412 participants who reported having at least one health care visit during the previous two years, 84% (10,404) agreed to medical record review and 7,528 signed consent forms. We obtained at least one record for 89% (6,712) of the respondents who consented, and we received 84% of the total records for which we had consent forms. Non-respondents were more likely to be female, older, and to have used health care services during the study period (p < .001). Development of quality indicators We developed process indicators for hypertensive quality of care by reviewing the scientific literature and clinical practice guidelines [ 15 ] pertaining to hypertensive care [ 16 , 17 ]. The indicators represented clinical processes across the spectrum of hypertensive care and were based closely on JNC-VI [ 15 ]. A diverse expert panel of nine physicians reviewed the indicators and supporting evidence using a modified Delphi method. They rated each indicator's feasibility and validity using a 9-point Likert scale. Indicators were accepted if their median validity score was 7 or higher and their median feasibility score was 4 or higher. The final 28 quality-of-care indicators included 1 screening indicator, 14 diagnostic indicators, 8 treatment indicators, and 5 follow-up indicators. Eight of the indicators were supported by randomized controlled trials and 20 by JNC-VI or other expert guidelines. The full list of indicators is presented in Table 1 . Table 1 Performance of recommended hypertensive care indicators Indicator Eligible (n) Adherence to Indicators (%) Standard Error (%) 1. Systolic and diastolic blood pressure should be measured on patients otherwise presenting for care at least once each year. 1,953 72 1.0 2. All patients with average blood pressures of Stage 1 or greater as determined on at least 3 separate visits should have a diagnosis of hypertension documented in the record. 823 73 1.8 3. Patients with a new diagnosis of Stage 1–3 hypertension should have at least 3 or more measurements on separate visits with a mean SBP > 140 or a mean DBP > 90. 185 21 3.6 4. Medication and substance abuse: Personal history of tobacco abuse, alcohol abuse, or taking of medications that may cause hypertension; 183 35 5.0 5. Physical examination: examination of the fundi 199 14 2.8 6. Examination of heart sounds 199 71 4.1 7. Examination of abdomen for bruits 199 49 4.6 8. Examination of peripheral arterial pulses 199 32 4.1 9. Examination of neurologic system 199 35 4.6 10. Initial laboratory tests should include at least 5 of the following: Urinalysis; 241 30 4.3 11. Serum, plasma, or blood glucose; 241 65 4.2 12. Serum potassium; 241 59 4.4 13. Serum creatinine; 241 62 4.2 14. Serum cholesterol; or 241 58 4.2 15. Serum triglyceride. 241 60 4.4 16. First-line treatment for patients in risk group HN-A or HN-B, is lifestyle modification. The medical record should indicate counseling for at least 1 of the following interventions prior to initiating pharmacotherapy: 27 31 10.2 - weight reduction if obese; - increased physical activity if sedentary; or - low sodium diet. 17. First-line treatment for patients with Stage 1A hypertension, is lifestyle modification. The medical record should indicate counseling for at least 1 of the following interventions prior to initiating pharmacotherapy: 25 25 10.4 - weight reduction if obese; - increased physical activity if sedentary; or - low sodium diet. 18. Treatment for Stage 1B and 1C, and Stages 2 and 3 hypertension should include lifestyle modification. The medical record should indicate counseling for at least 1 of the following interventions: 149 40 5.3 - weight reduction if obese; - increased physical activity if sedentary; or - low sodium diet. 19. Stage 1B hypertensives whose blood pressure remains Stage 1 after 6 months of lifestyle modification recommendation should be offered pharmacotherapy. 113 20 4.5 20. Stage 1A hypertensives whose blood pressure remains Stage 1 after 12 months of lifestyle modification recommendation should be offered pharmacotherapy. 22 14 7.6 21. Patients in any risk group with Stage 2–3 hypertension should be offered pharmacotherapy. 359 64 3.4 22. Patients in Risk group HN-C should be offered pharmacotherapy. 277 67 3.9 23. Patients in Risk group C with stage 1 hypertension should be offered pharmacotherapy. 332 75 2.9 24. Hypertensive patients should visit the provider at least once each year. 1,681 94 0.7 25. Newly diagnosed Stage 1 patients should be evaluated by the provider within 4 months of their initial visit. 111 76 5.1 26. Newly diagnosed Stage 2 patients should be evaluated by the provider within 2 months of their initial visit. 56 66 7.6 27. Newly diagnosed Stage 3 patients should be evaluated by the provider within 2 weeks of their initial visit. 18 33 12.9 28. Hypertensive patients with consistent average SBP > 140 or DBP > 90 over 6 months should have one of the following interventions recorded in the medical record: 853 77 1.8 - Change in dose or regimen of antihypertensives; or - Repeated education regarding lifestyle modifications. Overall 72 1.0 Explanation of staging system used in Table 1 Risk group A indicates no CAD risk factors or target organ damage or CAD. Risk group B indicates CAD risk factors, but no target organ damage or CAD or DM. Risk group C indicates target organ damage, DM or CAD. HN high-normal indicates 130–139 or 85–89. Stage 1 hypertension indicates 140–159 or 90–99. Stage 2 hypertension indicates 160–179 or 100–109. Stage 3 hypertension indicates ≥180 or ≥110. Data collection After a two-week training course, twenty nurse abstractors used a computer-based abstraction tool with branching logic to abstract from the medical records we collected the following data for the two-year study period: a diagnosis of hypertension; a diagnosis of co-morbid disease, including CAD, diabetes, and hyperlipidemia; BP readings; laboratory results, including serum creatinine, cholesterol, trigylcerides, sodium, potassium, and urinalyses; prescriptions for anti-hypertensive agents; and counseling for lifestyle modification. The Kappa statistic for inter-rater reliability for indicator eligibility was .75 and, given agreement on eligibility, the kappa statistic for the indicated care was .91. Definitions 86% of patients with a visit in the last 13 months of the study period had a blood pressure measured. Patients were included in the study if their provider noted a diagnosis of hypertension in their medical record or if they had BP readings greater than 140/90 on two occasions at least two weeks apart. However, patients were only classified as having a new diagnosis of hypertension if this was specifically noted in their medical records. Per JNC-VI, we defined Stage 1 hypertension as BP 140/90–159/99 and Stage 2 hypertension as BP > 160/100; we determined stage by averaging a patient's BP over the study period, regardless of notation of diagnosis or treatment regimen. We defined uncontrolled hypertension as a last systolic BP ≥ 140 or a last diastolic BP ≥ 90. Analytic methods We determined both individual and overall quality scores for each patient. We calculated individual scores by determining a patient's eligibility for an indicator and whether s/he received the indicated care. We calculated overall quality scores by adding the number of instances in which a patient was eligible for and received the indicated care and dividing this number by the total number of instances for which s/he was eligible for the indicated care; this score was expressed as a percentage. We defined optimal quality as patients having received all of the indicated care for which they were eligible. We adjusted the scores for non-response using multivariate models that weighted respondents to be representative of the population from which they were drawn. We used the bootstrap method to directly estimate standard errors for all of the individual indicator scores [ 18 ]. Statistical comparisons at the bi-variate level were reported as t-tests for continuous variables and chi-square tests for categorical discontinuous variables. A multivariate regression model was constructed to explain BP control using clinical and demographic variables available from the medical record. Results Of the 6,712 adult patients for whom we analyzed medical records1,953 (29%) had a diagnosis of hypertension noted in their medical record or repeated BP readings indicative of hypertension. Of these, 241 (12%) were newly diagnosed during the study period; 1,102 (57%) had hypertension noted in the medical record, but the diagnosis was not new; 610 (31%) had repeated BPs ≥ 140 systolic and/or 90 diastolic evidencing hypertension, but no diagnosis in the chart. The sample included 1,070 women (55%), 1,326 patients over age 50 (68%), and 918 (47%) patients with CAD, hyperlipidemia, or diabetes noted in their medical records. The average last systolic BP was 139.2 and the average last diastolic BP was 82.0. Of the 1,368 patients (70%) who were receiving pharmacological treatment for hypertension, 25% were prescribed beta-blockers, 37% diuretics, 30% calcium channel blockers, and 35% angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists. Patients with a diagnosis of hypertension noted in their records were more likely to have received pharmacotherapy during the study period than those with elevated BP readings but no notation of a diagnosis (88% vs. 31%, p < .0001). On average, patients were eligible for 3.8 indicators (range: 1–21 and visited their providers 13 times during the study period. Table 1 shows the performance of the 28 indicators among the sample. Indicator #1 had the largest number of eligible patients (n = 1,953); Indicator # 27 had the fewest number of eligible patients (n = 20). Indicator scores ranged from a low of 14% for Indicators #5 and #20 to a high of 94% for Indicator #24. Indicator #28 required a change in regimen in response to persistently elevated blood pressure; 80% of patients satisfied this requirement with a change in medication and 20% with repeated counseling on nonpharmacologic means of BP control. The mean patient overall quality score was 72%, and 57% of patients (1,113) received optimal quality of care (i.e., they received all recommended care for which they were eligible). The overall quality score was higher for patients with a diagnosis of hypertension noted in their chart (86% vs. 41%, p < .0001). The last BP reading for 42% (825) of patients indicated good control (<140/90). Table 2 shows the proportion of patients with well-controlled BP who received optimal and sub-optimal care by subgroup. Overall, patients with optimal quality care were more likely to have their BP under control (44.9% vs. 35.1%, p < .0006). This held true for all age groups, and for men, but was not statistically significant for women. Patients with optimal quality scores who did not have diabetes, CAD, or hyperlipidemia were more likely to have their BP controlled than those with sub-optimal scores. Table 2 Proportion of patients with well controlled BP by level of quality of care received Group N % <140/90 (Standard error) P-value Optimal quality Sub-optimal quality All 1,953 44.9 (2.0) 35.1 (2.0) .0006 Female 1,070 41.2 (2.5) 37.5 (2.7) .3209 Male 883 48.9 (2.8) 32.7 (3.0) .0001 Age <= 50 627 49.5 (3.4) 39.1 (3.0) .0234 Age > 50 1,326 43.4 (2.4) 32.2 (2.7) .0020 Diabetes 372 48.5 (3.7) 43.9 (5.4) .4797 No diabetes 1,581 44.0 (2.2) 33.5 (2.2) .0008 CAD 337 43.6 (3.9) 38.4 (6.3) .4835 No CAD 1,616 45.3 (2.3) 34.8 (2.1) .0007 Hyperlipidemia 604 47.4 (3.3) 39.7 (4.2) .1496 No hyperlipidemia 1,349 43.5 (2.4) 33.7 (2.3) .0033 CAD, DM, or hyperlipidemia 918 46.2 (2.7) 41.9 (3.5) .3237 Smoker 352 47.6 (4.7) 37.9 (4.1) .1218 Nonsmoker 1,601 44.4 (2.1) 34.3 (2.4) .0015 A logistic regression (not shown) revealed that the relationship between higher quality scores and BP control persisted when controlling for number of physician visits, baseline blood pressure control, gender, age, smoking status, and presence of CAD, diabetes, or hyperlipidemia. Other than quality, only baseline BP control was significantly associated with BP control at the end of the study. The relationship between quality and BP control also persisted when we restricted the study sample to those with at least two BP readings 12 months apart (n = 1,321). Likewise, there were no changes in direction or significance when we controlled for BP at 160/100 or used the continuous measures of last systolic or diastolic BP reading as the outcome. Figure 1 shows the relationship between quality scores and various demographic and clinical characteristics. There was no significant difference between quality for men and women; however, patients over age 50 received better care than their younger cohorts (76% vs. 63%, p = <.0001), as did patients with diabetes (77% vs. 71%, p = .0038), CAD (87% vs. 69%, p = <.0001), or hyperlipidemia (80% vs. 68%, p = <.0001). In addition, smoking was associated with lower quality of care (66% vs. 73%, p = .0005). Figure 1 Overall process quality scores by demographic and clinical subgroups Discussion Our data suggest that quality of care for hypertensive patients falls short of the ideal. Overall, patients received about 72% of recommended essential care processes, and 77% of patients with persistently elevated blood pressure had some change in therapy noted in the medical record over the course of two years. Though still concerning, these rates are higher than in some previous studies in more limited populations [ 9 , 19 ]. Some of the differences may be due to differences in the study population, and some may be due to more liberal definitions of what constitutes a change in therapy. We allowed longer time periods for the change to occur, and counted switching to different but possibly equipotent regimens and repeated nonpharmacologic interventions as therapeutic changes. Patients with additional risk factors for cardiovascular events were more likely to receive recommended care, suggesting that providers may be targeting patients at highest risk for hypertensive complications. Alternatively, these patients may have had better BP control because they were already engaged in treatment for other conditions. Smoking was the exception to this pattern. Smokers received lower quality of care even though they are at higher risk for cardiac complications from hypertension. This is particularly surprising because cigarette smoking is the only measured risk factor that directly and immediately affects BP, though its effect on long-term hypertension is unclear [ 20 ]. Further research is needed to identify possible explanations, but we hypothesize that physicians provide lower quality of care to smokers because they perceive them as unwilling to participate in their own care or less likely to comply with medical therapy. As in previous studies of hypertensives who have visited their physician, just under half of hypertensive patients had controlled BP. However, patients who received optimal care (i.e., they received all indicated care for which they were eligible) were more likely to have controlled BP at the end of the study, supporting the hypothesis that a broad range of hypertensive process quality is associated with better intermediate outcomes. The study was not powered to analyze which specific subcomponents of hypertensive care were most associated with BP control. However, because controlled hypertension is correlated with fewer myocardial infarctions and cerebrovascular accidents, it also seems likely that better overall care processes would eventually be associated with fewer complications from hypertensive care, although we could not directly test for this. This process-outcome relationship between hypertensive care and BP control was observed in almost all subgroups, but differences in quality were statistically significant primarily in patients without other cardiac risk factors [ 4 ]. Unfortunately, our study could not detect small differences in the relationship between quality of care and BP control within cardiac risk factor subgroups, and this is likely to partially explain this finding. However, for patients with CAD or diabetes, another potential explanation could be a "ceiling effect": diabetic patients received 77% of recommended care and patients with CAD received 87%, leaving little variation in quality to explain differences in BP control. In addition to poor process quality, there are other possible explanations for the high rates of uncontrolled BP observed in this study. Even among patients with at least one medical visit in the last two years, impaired access to care might be a factor, and this problem was likely worse in the 863 patients with no visits during the study period who were not included in the analysis [ 4 ]. Among those included in the study, however, impaired access was likely a small factor since the average number of provider visits in the two-year study period was 14 for patients with controlled BP and 12 for those with uncontrolled BP. In addition, hypertensive quality of care is no better in Canada despite the presence of universal health insurance in Canada and high prevalence of access problems in the U.S. [ 21 ], suggesting that sub-optimal clinical care processes are as important as access in explaining our results. Patient noncompliance has also long been recognized as an important predictor of poor BP control, but we did not measure it directly [ 22 ]. Our study is the first to examine the relationship between the quality of hypertensive care and outcomes among a national population; however, some study limitations exist beyond those inherent in retrospective medical record reviews. First, in constructing the indicators, our expert panel was instructed to rate indicators highly only if documentation of the process in the medical record was common and required for good quality care. Nonetheless, it is possible that documentation differences rather than true quality differences explain some of the observed variations in process quality or outcomes. A previous study found that differences in process scores were 10% lower for medical record abstraction compared to standardized patients with audiotapes of encounters [ 23 ]; thus, our study may underestimate actual quality of care by that amount. Second, we chose 140/90 as the threshold for poor BP control based on expert guidelines in force at the time of the study, but this threshold could potentially affect our results. However, sensitivity analyses using a higher threshold (160/100) and continuous distribution did not change our results. Later guidelines have suggested lower thresholds for diabetics; our findings remained consistent when we used 130/85 for this population. Third, providers' failure to measure the blood pressure could have caused us to erroneously exclude some patients from the analysis, though the vast majority of the studied patients had regular blood pressure measurement. Fourth, our response rate could have biased our results, particularly our estimates of overall quality. To account for this problem, we used standard techniques to adjust for non-response. Moreover, responders were more likely to be older and have chronic conditions than non-responders. Since we found higher quality of care among these subgroups, it is likely that our estimates represent upper bounds. Finally, our study does not take into account the lower threshold for pre-hypertension that was recently published in JNC-VII [ 5 ], since the care we evaluated was delivered prior to this recommendation. Future research will need to investigate whether the new standards are associated with better control. Quality assessment in hypertension is only useful if it is linked with efforts to improve care. There is some evidence that quality improvement (QI) programs can lead to BP control among hypertensive patients. Godley has recently showed that a QI program in a group-model managed care organization increased BP control from 37% to 49% [ 24 ]. Our results can help providers focus on the processes most likely to improve control and avoid adverse outcomes. Conclusions We found that the average hypertensive patient in the United States did not receive one in four essential care processes, and those with sub-optimal care had worse BP control. Our data indicate that improvements in processes of care may lead to better outcomes. While it appears that providers appropriately focus attention on patients with additional cardiovascular risk factors, they are under-treating other hypertensive patients who (as previous research would suggest) are nonetheless at increased risk for the same adverse outcomes. Future research should test if the application of routine measurement of hypertensive process quality in provider groups or plans leads to improved processes and BP control. Competing interests Drs. Asch, McGlynn, Adams, and Kerr declare they have no competing interests. Ms. Hiatt, Hicks, and DeCristofaro declare they have no competing interests. Drs. Chen and LaPuerta are employees and own shares of Bristol-Myers Squibb Company. Authors' contributions SA and BM served as principal investigators of this study and were involved in all aspects, as were EK and LH. PL and RC helped arrange partial funding and were involved in the analysis and writing of the manuscript. JA led the analysis, AD and JH were involved in supervising the data collection and participated in the analysis. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546215.xml
514547	Synergic effect of chronic hepatitis C infection and beta thalassemia major with marked hepatic iron overload on liver fibrosis: a retrospective cross-sectional study	Background Increased hepatic iron is assumed to potentiate progression towards liver fibrosis in chronic hepatitis C virus (HCV) infection. In this study we have evaluated the potentiating effect of marked hepatic iron overload and chronic HCV infection on hepatic fibrosis in thalassemic patients. Methods Liver biopsies of one group of patients with beta thalassemia major and chronic HCV infection (group 1) was compared with two groups of patients (groups 2&3) with either chronic HCV infection or thalassemia major, respectively (20 patients in each group). Necroinflammation, fibrosis, and iron overload were graded and compared. Results Stage of fibrosis in group 1 patients was significantly higher than the other two groups (p < 0.05). Necroinflammatory grade was significantly lower, but iron score was significantly higher in thalassemic patients (group 3) in comparison to groups 1 and 2 (p < 0.05). Conclusion Our results indicate that marked liver iron overload and HCV infection in thalassemic patients have potentiating effect on hepatic fibrogenesis.	Background Thalassemia major is an inherited disorder particularly common in people of Mediterranean, African, and South-east Asian ancestry. It is characterized by decreased production of beta chain of hemoglobin. Clinical features result from anemia, markedly expanded marrow space, and transfusional and absorptive iron overload. In these patients iron overload is often inevitable, especially when iron chelating agents are not used properly [ 1 ]. Hepatic iron overload leads to different degrees of liver fibrosis, the severity of which is closely correlated with the severity of liver iron overload [ 2 ]. The pattern of iron deposition seen in the initial stages of thalassemia major is preferentially sinusoidal with a more or less diffuse distribution within the acinus. With significant loading, hepatocytes, bile duct epithelia, and fibrous tissue of portal tracts or septa will also show iron deposition [ 3 ]. HCV is responsible for 80–90% of post-transfusional cases of hepatitis in patients who have received blood transfusion(s) prior to the introduction of routine blood products screening in 1990 [ 4 ]. More than 75% of HCV infections become chronic and up to 20–30% progress to cirrhosis [ 3 - 5 ]. Increased hepatic iron may potentiate progression towards liver fibrosis in chronic HCV infection, and may contribute to poor response to interferon therapy [ 3 , 6 , 7 ]. Acceleration of hepatic fibrosis in patients with combined hereditary hemochromatosis (HH) and chronic hepatitis C infection has also been shown [ 7 ]. Nevertheless, the pattern of iron deposition in HH is initially hepatocellular and different from that of thalassemia major. In this study we have evaluated the potentiating effect of marked hepatic iron overload and chronic HCV infection on hepatic fibrosis in thalassemic patients. To the best of our knowledge this synergic effect has been studied just in another study on bone marrow transplanted thalassemic patients [ 8 ]. Methods A retrospective cross-sectional study was performed on sixty patients in three different groups as outlined below: Group 1: Twenty patients (10 males, 10 females) with the diagnosis of thalassemia major and chronic HCV infection (BTM/CHI). The only risk factor of HCV infection in these patients was blood transfusion before 1990. Group 2: Twenty patients (13 males, 7 females) with chronic HCV infection (CHI). The route of infection and the duration of disease were not known in most of the patients. Group 3: Twenty patients (10 males, 10 females) with thalassemia major. Thalassemia major was diagnosed by appropriate clinical and laboratory findings, and confirmed by hemoglobin electrophoresis. All the thalassemic patients had received multiple blood transfusions since childhood, and only those with at least moderate hepatic iron overload (2/4 based on Marx and Sindram hepatic iron scoring [ 9 ]) were included. CHI was confirmed by positive anti-HCV (enzyme-linked immunosorbant assay), a positive HCV RNA by polymerase chain reaction and appropriate findings on liver biopsy. Patients with history of alcohol intake, smoking, positive HIV serology, positive HBs antigen or HBc antibody, and other liver diseases (e.g., autoimmune hepatitis, drug hepatotoxicity, Wilson's disease, alpha-1 antitrypsin deficiency, hereditary hemochromatosis) were excluded from all the three groups. All of the patients whose liver biopsies were submitted to the Central Pathology Department of Imam Khomeini Hospital (a Terhan University of Medical Sciences affiliated hospital) between April 2001 and January 2004 were retrieved. During this period, there were only 20 thalassemic patients with moderate to marked hepatic iron overload, so all of them were included in our study. Since the sex and age distribution of patients with thalassemia major and BTM/CHI were relatively the same, 20 patients with BTM/CHI and at least moderate hepatic iron overload were randomly selected from all the patients with this diagnosis. On the other hand, patients with CHI had a considerably higher age, so to match the age of the patients, we selected 20 of the youngest patients. For all the sixty patients, we had hematoxylin and eosin, Masson's trichrome and Perls' Prussian blue stains. All the slides were reviewed by a single pathologist (FAA) who was blind to the diagnoses. The necroinflammation, fibrosis, and iron deposition were scored using modified Hepatitis Activity Index (HAI) grading, modified HAI staging, and Marx/Sindram hepatic iron scoring systems, respectively [ 9 , 10 ]. Presence or absence of macrovesicular steatosis was also assessed. Statistical analysis The results are presented as mean value +/- standard deviation (for age), median and range values (for modified HAI grade, stage, and iron score) and percentages (for steatosis). Group comparisons were made using the two-tailed independent Student's t-test for age and two-sided Fisher's exact or Chi 2 tests for other variables with p < 0.05 considered to be significant. For each patient with BTM/CHI (doubly exposed cases), there were two different unexposed cases, i.e. thalassemic cases (unexposed to HCV) and CHI cases (unexposed to iron overload). Considering "disease" as higher stages of fibrosis (modified HAI stage ≥ 3), our guesstimate of the expected frequency of disease in unexposed and exposed patients were about 25% and 75%, respectively. So the sample size was calculated as about 20 patients for each group with confidence level of 95% and power of 80%. Results and Discussion The results of the study are summarized in table 1 . This study supports the synergic effects of CHI and marked hepatic iron deposition in thalassemic patients on liver fibrosis. The patients with BTM/CHI had higher stages of liver fibrosis in comparison to patients with either CHI or thalassemia major alone. The patients with BTM/CHI had higher stages of fibrosis despite lower scores for iron overload in comparison to thalassemic patients. The potentiating effect of hepatic iron overload and CHI has been shown in other studies [ 7 , 8 , 11 , 12 ]. The cause of iron overload in the majority of studies has been hereditary hemochromatosis or HFE mutations. Table 1 Demographic and histopathologic findings in the three groups Group 1 Group 2 Group 3 P value Group 1 vs. 2 Group 1 vs. 3 Age(years) * 21.55+/-4.74 29.7+/-9.16 18.7+/-4.45 0.001 0.057 Sex (M/F) 10/10 13/7 10/10 0.33 1 Modified Stage ‡ 3 (1–6) 2 (0–6) 2 (1–4) 0.02 0.01 Modified HAI grade ‡ 5 (1–8) 6 (2–11) 2 (0–6) 0.36 0.001 Periportal inflammation ‡ 1.5 (0–3) 2 (0–4) 0 (0–2) 0.77 0.004 Confluent Necrosis ‡ 0 (0–1) 0 (0–2) 0 (0–1) 0.13 0.091 Focal Necrosis ‡ 1 (0–2) 2 (1–2) 1 (0–2) 0.10 0.45 Portal Inflammation ‡ 2 (0–3) 2 (1–4) 1 (0–2) 0.87 0.002 Iron Score ‡ 3 (2–4) 0 (0–1) 4 (2–4) 0.000 0.024 Steatosis (%) 10 20 0 0.66 0.48 * mean+/-standard deviation ‡ Median (minimum-maximum) Group 1: Patients with beta thalassemia major and chronic HCV infection Group 2: Patients with chronic HCV infection Group 3: Patients with beta thalassemia major Angelucci et al showed for the first time the role of iron overload and HCV positivity as independent risk factors for hepatic fibrosis progression in thalassemic patients following successful bone marrow transplantation [ 8 ]. The Angelucci's study has a few advantages over our study: firstly, serial liver biopsies have been studied and the rate of liver fibrosis progression assessed. Secondly, hepatic iron concentration was used instead of scoring of stainable iron. Thirdly, two pathologists reviewed the slides independently. And finally, the sample size was much larger, but that study does not show whether the rate of hepatic fibrosis progression in BTM/CHI patients is greater than patients with only CHI. The potentiating effect of iron overload and HCV infection can be explained by the fact that both these agents produce oxidative stress in the liver [ 7 , 13 ]. In our study the grade of necroinflammation in BTM/CHI patients was not significantly different from CHI patients. Other studies have shown the same results [ 3 , 7 ]. Limitations of our study were that it was a retrospective study with limited number of patients in each group. The duration of HCV infection and genotype of virus were not known in our patients. The routes of infection in most of the CHI patients were not known and where probably different from our thalassemic patients. The age of our CHI group was significantly higher than the other two groups. Since older age at the time of infection is considered to be a risk factor for progression of chronic hepatitis C, the stage of fibrosis should have been higher in our CHI patients than BTM/CHI patients if iron had not had any potentiating effect on liver fibrosis. Our study was performed on thalassemic patients with moderate to severe liver iron deposition. At these stages of iron overload, the pattern of hepatic iron deposition in HH and thalassemic patients are nearly the same. So this study does not reveal the effect of only sinusoidal iron deposition on progression of fibrosis in HCV infected thalassemic patients. Conclusions Our results show that moderate to severe liver iron overload and chronic HCV infection in thalassemic patients have potentiating effect on hepatic fibrogenesis. So the proper use of chelating agents in HCV-infected thalassemic patients seems to be of great importance in delaying progression of the liver disease. The presence of liver siderosis has been shown to be related to poor response to interferon alpha (IFN) in non-thalassemic patients [ 3 , 6 , 7 , 12 , 14 , 15 ]; hence one can expect a poor response to IFN therapy because of transfusion related siderosis in thalassemic patients. However studies have shown that in thalassemic subjects, there is a promising response to IFN therapy (as high as 50% sustained response in some series) [ 16 , 17 ]. Perhaps in these patients (BTM/CHI), the therapeutic protocol for chronic HCV infection should differ from those without significant iron overload, but further studies are needed to confirm or refute these suggestions. Competing interests None declared. Authors' contributions FAA reviewed all of the slides, participated in study design and statistical analysis of results, and prepared the manuscript. MRFO assisted in collection and entering of the data, cooperated in reviewing slides, statistical analysis of results, and preparing the manuscript. MN was the clinical consultant. GI reviewed some of the problematic slides. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514547.xml
545046	In-situ and invasive carcinoma within a phyllodes tumor associated with lymph node metastases	Background Phyllodes tumors (cystosarcoma phyllodes) are uncommon lesions in the female breast. Rarely, the occurrence of carcinoma within a phyllodes tumor has been reported in the literature, but has never been associated with lymph node metastases. Case presentation A 26-year-old woman presented with a firm, mobile, non-tender mass in the left breast and palpable lymph nodes in the left axilla. The excised lesion appeared well circumscribed and lobulated, with variable fleshy and firm areas. Microscopic examination showed a circumscribed fibroepithelial lesion with a well developed leaf-like architecture, in keeping with a benign phyllodes tumor. The epithelial component showed extensive high grade ductal carcinoma in-situ (DCIS) and invasive carcinoma of no special type, located entirely within the phyllodes tumor. Subsequent axillary lymph node dissection revealed metastatic carcinoma in four lymph nodes. Conclusions Although rare, phyllodes tumors may harbor DCIS and invasive carcinoma, with potential for lymph node metastasis.	Background Phyllodes tumors constitute less than 1% of breast tumors and 2–3% of fibroepithelial breast tumors [ 1 ]. They usually occur in middle-aged to elderly women but can occur at any age. No single feature is reliable in predicting clinical behavior of phyllodes tumors. Several histological parameters should be evaluated, including stromal cellularity, atypia, mitoses, stromal overgrowth, infiltrative borders, and presence or absence of necrosis [ 1 , 2 ]. A mitotic rate of less than 5 per 10 high power field (HPF) suggests benign behavior, while a mitotic figure rate of more than 10 per 10 HPF suggests malignant potential [ 2 , 3 ]. Most of these tumors are benign, but up to 30% show malignant stroma [ 4 ]. Metastases, usually hematogenous rather than lymphatic, have been reported to occur at a rate of 13% in 10 years for malignant phyllodes tumors [ 4 ]. As malignant phyllodes tumors usually spread by a hematogenous rather than a lymphatic route, axillary lymph node dissection is generally not recommended. Importantly, local recurrences are common even in benign tumors and are seen in up to 8% in 10 years [ 4 ]. We describe a case of in-situ and invasive carcinoma occurring within a benign phyllodes tumor in a 26-year-old woman, which, to our knowledge, is the youngest presenting age for this combination tumor. We also report the first case of this combination tumor with documented lymph node metastases. Our case highlights the importance of assessing phyllodes tumors for concurrent carcinomatous involvement, as this may affect both the need for axillary lymph node examination and subsequent treatment options. Case presentation A 26-year-old woman presented with a 1 month history of a palpable mass in her left breast. Her mother died at the age of 31 years of breast cancer. Examination revealed a 2 cm firm and mobile mass in the lower outer quadrant of the left breast. The left axilla contained two enlarged palpable lymph nodes. Mammography showed a 2 cm oval density that was homogenous with sharp margins. Ultrasound characterized the mass as solid but with a complex internal architecture. Fine needle aspiration of the mass was performed. It was positive for carcinoma, showing a very cellular sample with many malignant epithelial cells in cohesive groups, as well as lying singly in a background of necrosis and inflammation. The surgeon subsequently performed an excisional biopsy. Grossly, the excised mass measured 3.3 cm × 2.3 cm × 2.0 cm and appeared well circumscribed. The cut surface was grey and lobulated with variable fleshy and firm areas. Histological examination revealed a well-circumscribed biphasic neoplasm, consisting of proliferating stroma and compressed glands, displaying a well developed leaf-like architecture. The stroma was moderately cellular, mildly vascular, and lacked cytological atypia, mitoses, and stromal overgrowth. The features were consistent with a benign phyllodes tumor (figures 1 & 2 ). More importantly, the epithelial component of the phyllodes tumor showed extensive ductal carcinoma in-situ , with multiple foci of invasive mammary carcinoma (figure 3 ). The DCIS was of high nuclear grade, cribriform and micropapillary architecture, with (comedo-type) necrosis (figure 4 ). The invasive carcinoma was of no special type, SBR Grade III/III (Score 8/9). Multiple tumor emboli were present within endothelial-lined spaces adjacent to the tumor, but the surrounding breast was otherwise unremarkable. Although the carcinomatous component was confined to the phyllodes tumor, the lesion was <1 mm from the nearest resection margin. Immunohistochemistry using smooth muscle myosin heavy chain and muscle specific actin demonstrated absence of the myoepithelial cell layer within areas of invasive carcinoma, and focal loss within areas of DCIS. There was intensely positive staining within 80% of tumor cells for estrogen and progesterone receptors. Figure 1 Low power photomicrograph of the phyllodes tumor: Histologically, the neoplasm was biphasic, with proliferating stroma, compressed glands, and a well developed leaf-like architecture (H&E, original magnification 40×). Figure 2 High power photomicrograph of the phyllodes tumor: The stroma lacked cellular atypia and mitoses, consistent with a benign phyllodes tumor (H&E, original magnification 400×). Figure 3 Photomicrograph of the carcinomatous component: The epithelial component showed DCIS and invasive mammary carcinoma (H&E, original magnification 100×). Figure 4 Photomicrograph of the DCIS patterns: The DCIS showed cribriform and micropapillary architecture, with necrosis (H&E, original magnification 100×). The patient went on to have a left axillary lymph node dissection and subsequent pathological examination revealed 4 of 13 nodes positive for metastatic adenocarcinoma. She received adjuvant CEF (Cyclophosphamide, Epirubicin, 5-Fluorouracil) chemotherapy and underwent further local excision to achieve wider margins; pathological examination was negative for residual tumor. She subsequently received local radiation and is currently undergoing tamoxifen therapy. The patient is presently alive and disease free, nearly three years after presentation. Discussion The epithelial component of phyllodes tumors (whether benign or malignant) may show a range of metaplastic (apocrine, squamous) and proliferative changes [ 5 , 6 ]. However, carcinoma arising within a phyllodes tumor is decidedly rare, with less than 30 reported cases in the literature. The age of the previously reported patients with coexistent carcinoma and phyllodes tumor ranged from 31–80 years, with most of the patients in the 5th or 6th decades [ 7 , 8 ]. This case represents the youngest age at presentation with this combination tumor. The reported subtypes include in-situ and invasive lobular and ductal (no special type) carcinoma, tubular carcinoma and squamous carcinoma [ 2 - 7 , 9 - 13 ]. Some authors have noticed greater atypia in epithelial components of recurrent phyllodes tumors. Squamous carcinoma, typically rare in the breast, seems to occur more often in phyllodes tumors, compared to patients without phyllodes tumors [ 10 ]. The prognosis for cases of carcinoma within phyllodes tumors is generally favorable, with no deaths yet reported [ 9 , 10 ]. The stroma is benign in the majority and cancer detection often occurs early because the patient presents with a rapidly enlarging mass [ 10 ]. Our case appears to represent the first documentation of lymph node spread of the carcinomatous component from within a phyllodes tumor. Axillary lymph node dissection is not part of the standard treatment for phyllodes tumors as lymph node spread is rare [ 14 ]. Several authors have suggested treating carcinoma within phyllodes tumors according to the carcinomatous component [ 9 , 14 ]. Our case supports this view, particularly with respect to the need for assessment of axillary lymph node status in determining prognosis and treatment. Conclusions Invasive carcinoma occurring within a phyllodes tumor is rare; occurrence of axillary lymph node metastasis is even rarer. Besides the stromal component, the epithelial component of phyllodes tumors should also be examined in detail, as it may harbor an in situ or invasive carcinoma. Competing Interests The authors declare that they have no competing interests. Authors' Contributions JRP prepared the manuscript. CA and ABT were involved with initial diagnosis of the pathologic specimen and were involved with manuscript editing. FO provided consultation for the final diagnostic interpretation and was involved with manuscript editing. JR provided patient history, obtained patient consent and helped with manuscript editing. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545046.xml
544833	Identification of a three-gene expression signature of poor-prognosis breast carcinoma	Background The clinical course of breast cancer is difficult to predict on the basis of established clinical and pathological prognostic criteria. Given the genetic complexity of breast carcinomas, it is not surprising that correlations with individual genetic abnormalities have also been disappointing. The use of gene expression profiles could result in more accurate and objective prognostication. Results To this end, we used real-time quantitative RT-PCR assays to quantify the mRNA expression of a large panel (n = 47) of genes previously identified as candidate prognostic molecular markers in a series of 100 ERα-positive breast tumor samples from patients with known long-term follow-up. We identified a three-gene expression signature ( BRCA2 , DNMT3B and CCNE1 ) as an independent prognostic marker (P = 0.007 by univariate analysis; P = 0.006 by multivariate analysis). This "poor prognosis" signature was then tested on an independent panel of ERα-positive breast tumors from a well-defined cohort of 104 postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone: although this "poor prognosis" signature was associated with shorter relapse-free survival in univariate analysis (P = 0.029), it did not persist as an independent prognostic factor in multivariate analysis (P = 0.27). Conclusion Our results confirm the value of gene expression signatures in predicting the outcome of breast cancer.	Background Breast carcinoma is the most common female cancer and is showing an alarming year-on-year increase. Most patients do not die as a result of the primary tumor but from metastatic invasion. The mean 5-year relapse-free survival rate is about 60% overall, but differs significantly between patients with forms that rapidly metastasize and those with less aggressive forms. Current clinical, pathological and biological parameters, i.e. age, menopausal status, lymph-node status, macroscopic tumor size, histological grade and estrogen receptor status, fail to accurately predict clinical behavior. Breast cancer initiation and progression is a process involving multiple molecular alterations, many of which are reflected by changes in gene expression in malignant cells. Many clinical studies have attempted to identify correlations between altered expression of individual genes and breast cancer outcome, but often with contradictory results. Examples of such genes include ERBB2 , CCDN1 , MYC , UPA and PAI1 [ 1 - 3 ]. It is thus likely that these genes have limited predictive power when considered in isolation, but that their clinical relevance may be increased when several genes are considered together. The recent development of effective tools for monitoring gene expression on a large scale is providing new insights into the involvement of gene networks and regulatory pathways in various tumor processes [ 4 ]. It has also led to the discovery of new diagnostic and prognostic indicators, and to the identification of new molecular targets for drug development [ 5 ]. These tools include cDNA microarrays, which can be used to explore the expression of thousands of genes at a time, and real-time RT-PCR assays for more accurate and quantitative studies of the expression of a smaller number of selected candidate genes. In this study, we used real-time quantitative RT-PCR assays to quantify the mRNA expression of 47 candidate prognostic molecular markers in a series of 100 ERα-positive breast tumor samples. We identified a three-gene expression signature ( BRCA2 , DNMT3B and CCNE1 ) associated with poor clinical outcome. We then tested this "poor prognosis" signature on an independent panel of ERα-positive breast tumor samples from a well-defined cohort of 104 postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone with known long-term follow-up. Materials and Methods Patients and samples We analyzed samples from two series of women with primary unilateral ERα-positive breast carcinoma. ERα-positive status was determined at both the protein level by the Dextran-coated charcoal method until 1988 and enzymatic immuno-assay thereafter, and at the mRNA level by real-time quantitative RT-PCR assay [ 6 ]. The first series consisted of 100 women whose breast tumors were excised at Centre René Huguenin from 1977 to 1987. The patients (mean age 58.1 years, range 34–91) were pre- or post-menopausal (37 and 63 patients, respectively). Sixty patients received adjuvant therapy, consisting of chemotherapy alone in 14 cases, hormone therapy alone in 15 cases, and both treatments in 31 cases. The standard prognostic factors are presented in Table 1 . The median follow-up was 9.3 years (range 1.4–16.2 years). Thirty-seven patients relapsed within 10 years after surgery. The first relapse events consisted of local and/or regional recurrences in 11 patients, metastases in 22 patients, and both events in four patients. Table 1 Characteristics of the first series of 100 ERα-positive breast tumor patients, and relation to RFS RFS Number of patients Number of events (%) a P value b Age NS (0.68) ≤50 32 11 (34.3) >50 68 26 (38.2) SBR histological grade c NS (0.14) I 16 3 (18.7) II 51 21 (41.1) III 26 13 (50.0) Lymph node status 0.042 Node-negative 34 7 (20.5) Node-positive 66 30 (45.4) Macroscopic tumor size d NS (0.97) ≤30 mm 69 26 (37.6) >30 mm 24 10 (41.6) a : First relapses (local and/or regional recurrences, and/or metastases). b : Log-rank test. NS, not significant. c : Scarff Bloom Richardson classification. Information available for 93 patients. d : Information available for 93 patients. The second series consisted of 104 post-menopausal women whose breast tumors were excised at Centre René Huguenin from 1980 to 1994. The patients (mean age 70.9 years, range 54–86) all received post-operative adjuvant hormone therapy consisting of tamoxifen (20 mg daily for 3–5 years) and no other treatment. The standard prognostic factors are reported in Table 2 . The median follow-up was 5.9 years (range 1.4–18.1 years). Thirty-one patients relapsed within 10 years after surgery. The first relapse events consisted of local and/or regional recurrences in five patients, metastases in 24 patients, and both events in two patients. Table 2 Characteristics of the second series of 104 ERα-positive postmenopausal breast tumor patients, and relation to RFS RFS Number of patients Number of events (%) a P value b Age NS (0.92) ≤70 52 17 (32.6) >70 52 14 (26.9) SBR histological grade c 0.0005 I 13 0 II 67 17 (25.3) III 23 13 (56.5) Lymph node status NS (0.17) Node-negative 17 2 (11.7) Node-positive 87 29 (33.3) Macroscopic tumor size d 0.015 ≤30 mm 71 16 (22.5) >30 mm 31 14 (45.1) a : First relapses (local and/or regional recurrences, and/or metastases) b : Log-rank test. NS, not significant. c : Scarff Bloom Richardson classification. Information available for 103 patients. d : Information available for 102 patients. Complete clinical, histological and biological information was available for the two series of breast cancer patients; no radiotherapy or chemotherapy was given before surgery, and full follow-up took place at Centre René Huguenin. The histological type of the tumor and the number of positive axillary nodes were established at the time of surgery. The malignancy of infiltrating carcinomas was scored according to Scarff Bloom and Richardson's (SBR) histoprognostic system. Both series of tumor samples were placed in liquid nitrogen until total RNA extraction immediately following surgery. Real-time RT-PCR (1) Theoretical basis Quantitative values are obtained from the cycle number (Ct value) at which the increase in fluorescent signal associated with an exponential growth of PCR products starts to be detected by the laser detector of the ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, CA) using the PE Biosystems analysis software according to the manufacturer's manuals. The precise amount of total RNA added to each reaction (based on optical density) and its quality (i.e. lack of extensive degradation) are both difficult to assess. We therefore also quantified transcripts of the gene TBP (Genbank accession NM_003194) encoding for the TATA box-binding protein (a component of the DNA-binding protein complex TFIID) as an endogeneous RNA control, and normalized each sample on the basis of its TBP content. Results, expressed as N-fold differences in target gene expression relative to the TBP gene, termed "N target ", were determined by the formula: N target = 2 ΔCt sample , where ΔCt value of the sample was determined by subtracting the average Ct value of the target gene from the average Ct value of the TBP gene. The N target values of the samples were subsequently normalized such that the N target value of the tumor sample which contained the smallest amount of target gene mRNA in each tumor series would equal a value of 1. (2) Primers and probes Primers and probes for TBP and the 47 target genes were chosen with the assistance of the computer programs Oligo 5.0 (National Biosciences, Plymouth, MN). We conducted searches in dbEST, htgs and nr databases to confirm the total gene specificity of the nucleotide sequences chosen for the primers and probes, and the absence of single nucleotide polymorphisms. In particular, the primer pairs were selected to be unique when compared with the sequences of the closely related family member genes or of corresponding retropseudogenes. To avoid amplification of contaminating genomic DNA, one of the two primers or the probe was placed at the junction between two exons. Agarose gel electrophoresis allowed us to verify the specificity of PCR amplicons. The list of the 47 target genes tested in this study is indicated in Table 3 . Table 3 List of the 47 target genes selected Genes a Genbank accession number Chromosomal location Description AR NM_000044 Xq11.2-q12 Androgen receptor AREG NM_001657 4q13-q21 Amphiregulin ARHC/RhoC NM_175744 1p13.1 Ras homolog gene family, member C BCL2 NM_000633 18q21.3 B-cell CLL/lymphoma 2 BRCA1 NM_007294 17q21 Breast cancer 1, early onset BRCA2 NM_000059 13q12.3 Breast cancer 2, early onset CAV1 NM_001753 7q31.1 Caveolin 1 CCND1 NM_053056 11q13 Cyclin D1 CCNE1 NM_001238 19q12 Cyclin E1 CD44 NM_000610 11p13 CD44 antigen CDH1 NM_004360 16q22.1 Cadherin 1 (E-cadherin) CGA NM_000735 6q12-q21 Glycoprotein hormones, alpha polypeptide CGB NM_000737 19q13.32 Chorionic gonadotropin, beta polypeptide CP/Ceruloplasmin NM_000096 3q23-q25 Ceruloplasmin CXCL12 NM_000609 10q11.1 Chemokine (C-X-C motif) ligand 12 CXCR4 NM_003467 2q21 Chemokine (C-X-C motif) receptor 4 DNMT3B NM_006892 20q11.2 DNA (cytosine-5-)-methyltransferase 3 beta EGFR/ERBB1 NM_005228 7p12 Epidermal growth factor receptor ERBB2 NM_004448 17q21.1 ErbB2 ERBB3 NM_001982 12q13 ErbB3 ERBB4 NM_005235 2q33.3-q34 ErbB4 ESR1/ERα NM_000125 6q25.1 Estrogen receptor 1 (alpha) ESR2/ERβ NM_001437 14q Estrogen receptor 2 (beta) ETV4/PEA3/E1AF NM_001986 17q21 Ets variant gene 4 HAS2 NM-005328 8q24.12 Hyaluronan synthase 2 HMMR/RHAMM NM_012484 5q33.2-qter Hyaluronan-mediated mobility receptor KRT19 NM_002276 17q21.2 Keratin 19 MKI67 NM_002417 10q25-qter Antigen identified by monoclonal antibody Ki-67 MYC NM_002467 8q24.12-q24.13 c-myc oncogene p14/ARF NM_058195 9p21 Alternative reading frame p14 (p14ARF) p15/CDKN2B NM_004936 9p21 Cyclin-dependent kinase inhibitor 2B (p15 CDK inhibitor) p16/CDKN2A NM_000077 9p21 Cyclin-dependent kinase inhibitor 2A (p16 CDK inhibitor) PGR/PR NM_000926 11q22-q23 Progesterone receptor PLAU/UPA NM_002658 10q24 Plasminogen activator, urokinase PTGS2/COX2 NM_000963 1q25.2-q25.3 Prostaglandin-endoperoxide synthase 2 PTTG1/Securin NM_004219 5q35.1 Pituitary tumor-transforming 1 RB1 NM_000321 13q14.2 Retinoblastoma 1 SERPINB2/PAI2 NM_002575 18q21.3 Plasminogen activator inhibitor type 2 SERPINB5/Maspin NM_002639 18q21.3 Maspin SERPINE1/PAI1 NM_000602 7q21.3-q22 Plasminogen activator inhibitor type 1 SPP1/Osteopontin NM_000582 4q21-q25 Secreted phosphoprotein 1 SRC NM_005417 20q12-q13 c-src oncogene TERT NM_003219 5p15.33 Telomerase reverse transcriptase TFF1/pS2 NM_003225 21q22.3 Trefoil factor 1 TIAM1 NM_003253 21q22.11 T-cell lymphoma invasion and metastasis 1 TOP2A NM_001067 17q21-q22 Topoisomerase (DNA) II alpha 170 kDa XLKD1/LYVE-1 NM_006691 11p15 Extracellular link domain containing 1 a LocusLink symbol (3) RNA extraction Total RNA was extracted from frozen tumor samples by using the acid-phenol guanidinium method. The quality of the RNA samples was determined by electrophoresis through agarose gels and staining with ethidium bromide, and the 18S and 28S RNA bands were visualized under ultraviolet light. (4) cDNA Synthesis Reverse transcription of total RNA was done in a final volume of 20 μL containing 1X RT buffer (500 μM each dNTP, 3 mM MgCl2, 75 mM KCl, 50 mM Tris-HCl pH 8.3), 20 units of RNasin RNase inhibitor (Promega, Madison, WI), 10 mM DDT, 100 units of Superscript II RNase H- reverse transcriptase (Invitrogen, Cergy Pontoise, France), 3 μM random hexamers (Pharmacia, Uppsala, Sweden) and 1 μg of total RNA. The samples were incubated at 20°C for 10 min and 42°C for 30 min, and reverse transcriptase was inactivated by heating at 99°C for 5 min and cooling at 5°C for 5 min. (5) PCR amplification All PCR reactions were performed using a ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems). PCR was performed using either the TaqMan ® PCR Core Reagents kit or the SYBR ® Green PCR Core Reagents kit (Perkin-Elmer Applied Biosystems). The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min and 50 cycles at 95°C for 15 s and 65°C for 1 min. Statistical Analysis The distributions of the gene mRNA levels were characterized by their median values and ranges. Relationships between mRNA levels of the different target genes and comparison between the target gene mRNA levels and the clinical parameters were estimated using nonparametric tests: the Mann-Whitney U test (link between 1 qualitative parameter and 1 quantitative parameter) and the Spearman rank correlation test (link between 2 quantitative parameters). Differences between the two populations were judged significant at confidence levels greater than 95% (p < 0.05). To visualize the efficacy of a molecular marker to discriminate two populations (in the absence of an arbitrary cutoff value), we summarized the data in a ROC (receiver operating characteristic) curve [ 7 ]. This curve plots the sensibility (true positives) on the Y axis against 1 – the specificity (false positives) on the X axis, considering each value as a possible cutoff value. The AUC (area under curves) was calculated as a single measure for the discriminate efficacy of a molecular marker. When a molecular marker has no discriminative value, the ROC curve will lie close to the diagonal and the AUC is close to 0.5. When a test has strong discriminative value, the ROC curve will move up to the upper left-hand corner (or to the lower right-hand corner) and the AUC will be close to 1.0 (or 0). Hierarchical clustering was performed using the GenANOVA software [ 8 ]. Relapse-free survival (RFS) was determined as the interval between diagnosis and detection of the first relapse (local and/or regional recurrences, and/or metastases). Survival distributions were estimated by the Kaplan-Meier method [ 9 ], and the significance of differences between survival rates was ascertained using the log-rank test [ 10 ]. Cox's proportional hazards regression model [ 11 ] was used to assess prognostic significance. Results mRNA expression of 47 genes in 100 ERα-positive breast tumors The results for the 47 genes are summarized in table 4 , with medians and ranges of mRNA levels in patients who relapsed (n = 37) and those who did not (n = 63). Table 4 Relationships between the prognostic (+/- relapses) and the mRNA levels of the 47 selected genes in 100 ERα-positive breast tumors GENES Tumors without relapses (n = 63) Tumors with relapses (n = 37) P a ROC-AUC b BRCA2 4.6 (1.0–12.4) c 7.1 (1.9–18.8) 0.0011 0.696 (0.59–0.80) d DNMT3B 3.0 (1.0–13.6) 4.6 (1.2–17.4) 0.0015 0.690 (0.58–0.80) CCNE1 6.2 (1.0–36.9) 8.9 (3.1–82.5) 0.0038 0.674 (0.57–0.78) HMMR/RHAMM 18.9 (1.0–163.5) 30.1 (3.8–186.5) 0.0068 0.663 (0.55–0.77) MKI67 9.1 (1.0–49.8) 14.4 (1.7–54.9) 0.016 0.645 (0.53–0.75) TERT 18.7 (1.0–121.9) 22.1 (1.8–135.8) 0.049 0.618 (0.50–0.73) TOP2A 40.9 (1.0–306) 55.6 (6.1–317) NS 0.605 (0.49–0.72) PLAU/UPA 4.6 (1.0–36.4) 5.8 (1.4–34.0) NS 0.588 (0.47–0.70) CGB 4.2 (1.0–31.2) 6.4 (1.4–32.8) NS 0.579 (0.46–0.70) ERBB2 14.0 (1.0–175) 16.3 (4.2–179.8) NS 0.573 (0.46–0.69) BRCA1 11.9 (1.0–44.5) 14.3 (1.8–62.5) NS 0.569 (0.45–0.69) CXCR4 6.5 (1.0–40.5) 7.5 (1.5–71.5) NS 0.569 (0.45–0.68) PTTG1/Securin 1.9 (1.0–26.9) 1.9 (1.2–33.1) NS 0.566 (0.45–0.68) SRC 2.6 (1.0–4.3) 2.9 (1.4–10.2) NS 0.561 (0.45–0.67) p16/CDKN2A 3.4 (1.0–107.4) 4.4 (1.1–136.7) NS 0.560 (0.44–0.68) AREG 89.3 (1.0–5667) 110.1 (3.1–3301) NS 0.555 (0.44–0.67) SERPINE1/PAI1 3.8 (1.0–21.4) 4.5 (1.3–21.8) NS 0.554 (0.44–0.67) ERBB3 2.6 (1.0–10.7) 3.3 (1.2–13.4) NS 0.552 (0.44–0.67) SERPINB5/Maspin 12.6 (1.0–321) 16.4 (1.0–718) NS 0.551 (0.43–0.67) CDH1 11.3 (1.0–32.6) 13.9 (1.5–33.3) NS 0.549 (0.43–0.67) p15/CDKN2B 3.5 (1.0–16.2) 4.2 (1.0–34.9) NS 0.548 (0.43–0.67) SPP1/Osteopontin 43.3 (1.0–1403) 56.8 (2.1–941) NS 0.548 (0.42–0.68) ETV4/PEA3/E1AF 5.1 (1.0–49.3) 6.9 (1.8–62.0) NS 0.545 (0.43–0.66) CP/Ceruloplasmin 33.5 (1.0–9815) 81.5 (1.0–33943) NS 0.545 (0.42–0.67) SERPINB2/PAI2 13.0 (1.0–498) 15.3 (1.0–1652) NS 0.535 (0.42–0.65) TIAM1 13.6 (1.0–55.9) 13.3 (3.9–83.2) NS 0.526 (0.41–0.64) RB1 4.2 (1.0–7.4) 4.3 (1.5–7.7) NS 0.520 (0.40–0.64) AR 54.2 (1.0–219) 64.8 (1.0–211) NS 0.518 (0.40–0.64) HAS2 6.5 (1.0–40.8) 6.4 (1.4–31.9) NS 0.516 (0.40–0.63) TFF1/pS2 1772 (1.0–138 545) 1783 (3–55 878) NS 0.509 (0.39–0.62) ESR2/ERβ 28.2 (1.0–368) 25.3 (1.4–219) NS 0.500 (0.38–0.62) ERBB4 141 (1.0–1489) 143 (2.1–1062) NS 0.483 (0.37–0.60) KRT19 14.4 (1.6–99.1) 10.8 (1.0–57.1) NS 0.482 (0.36–0.60) ESR1/ERα 25.5 (1.0–508) 21.7 (1.2–498) NS 0.479 (0.36–0.60) CXCL12 12.1 (1.3–36.1) 9.6 (1.0–30.5) NS 0.464 (0.35–0.58) MYC 8.1 (1.0–35.5) 7.5 (1.0–51.2) NS 0.464 (0.35–0.58) EGFR/ERBB1 8.3 (1.2–108) 6.2 (1.0–66.8) NS 0.462 (0.34–0.58) ARHC/RhoC 6.9 (1.0–192) 6.3 (1.0–17.2) NS 0.458 (0.34–0.58) p14/ARF 4.9 (1.4–68.1) 4.4 (1.0–61.2) NS 0.457 (0.34–0.57) XLKD1/LYVE-1 4.5 (1.4–10.9) 3.7 (1.0–10.7) NS 0.448 (0.33–0.57) CD44 3.1 (1.2–9.6) 2.7 (1.0–8.4) NS 0.440 (0.32–0.56) CGA 17.6 (1.0–16 552) 6.4 (1.0–5 836) NS 0.425 (0.31–0.54) CAV1 7.4 (1.1–30.7) 5.6 (1.0–26.6) NS 0.422 (0.31–0.54) BCL2 4.9 (1.2–13.3) 3.2 (1.0–11.8) NS 0.415 (0.30–0.53) PGR/PR 277 (1.0–8 034) 97 (1.0–4 551) NS 0.412 (0.29–0.53) PTGS2/COX2 4.6 (1.0–154) 3.0 (1.0–14.8) NS 0.397 (0.28–0.51) CCND1 6.3 (1.2–45.3) 4.0 (1.0–21.3) 0.042 0.378 (0.26–0.50) a: P value, Mann-Whitney U test ; NS, not significant b: ROC ( Receiver Operating Characteristics ) – AUC ( Area Under Curve ) analysis c: Median (range) of gene mRNA levels d: AUC value (95% confidence interval) Seven genes showed significantly different expression according to relapse status (P < 0.05), namely BRCA2 , DNMT3B , CCNE1 , HMMR/RHAMM , MKI67 , TERT and CCND1 . The prognostic performance of these 7 genes was also assessed using ROC-AUC analysis. BRCA2 emerged as the most discriminatory marker of relapse status (ROC-AUC, 0.696). The mRNA expression of this gene, as well as DNMT3B , CCNE1 , HMMR/RHAMM , MKI67 and TERT , was higher in patients who relapsed than in patients who did not relapse, while only CCND1 mRNA expression was lower in patients who relapsed. On hierarchically clustering the tumor samples according to the expression of the three most discriminatory genes i.e. the genes with the highest ROC-AUC values ( BRCA2 , DNMT3B and CCNE1 , ROC-AUC: 0.696, 0.690 and 0.674, respectively), the patient population fell into two subgroups (65 and 35 subjects, respectively) with significantly different relapse-free survival curves (log-rank test, P = 0.007; Figure 1A ) (5-year RFS rate 66.9% ± 8.1 versus 83.9% ± 4.6; 10-year RFS rate 41.0% ± 8.7 versus 67.0% ± 6.6). Figure 1 Relationship between RFS and the three-gene expression signature in the initial series of 100 ERα-positive breast tumor samples (A) and in an independent series of 104 ERα-positive postmenopausal breast tumor samples (B). The prognostic value of a two-gene expression signature based on only BRCA2 and DNMT3B was lower than that of the three-gene expression signature. The addition of HMMR/RHAMM and/or MKI67 to the three-gene signature provided no additional prognostic value. Using a Cox proportional hazards model, we also assessed the prognostic value, for RFS, of parameters that were significant or near-significant (P < 0.2) in univariate analysis, i.e. SBR grade, lymph-node status (Table 1 ) and the three-gene expression signature (Figure 1A ). Only the prognostic significance of the three-gene expression signature persisted [P = 0.006; regression coefficient = 0.86; relative risk (95% confidence interval) = 2.37 (1.27–4.43)]. The prognostic significance of these three parameters for RFS, calculated in terms of the relative risk, did not change after adjustment for age and macroscopic tumor size (data not shown). Validation of the three-gene expression signature in an independent series of 104 ERα-positive postmenopausal breast tumor samples The results for each of the three genes are summarized in table 5 , with medians and ranges of mRNA levels in the 31 patients who relapsed and the 73 patients who did not relapse, as well as ROC-AUC values. As in the initial tumor series, BRCA2 , DNMT3B and CCNE1 mRNA levels were significantly higher in patients who relapsed than in those who did not relapse. Table 5 Relationships between the prognostic (+/- relapses) and the mRNA levels of BRCA2 , DNMT3B and CCNE1 in 104 ERα-positive postmenopausal breast tumors GENES Tumors without relapses (n = 73) Tumors with relapses (n = 31) P a ROC-AUC b BRCA2 4.7 (1.0–23.6 c 7.5 (1.4–38.4) 0.0018 0.694 (0.58–0.80) d DNMT3B 3.6 (1.0–27.8) 6.2 (2.3–74.1) 0.00052 0.716 (0.61–0.82) CCNE1 6.4 (1.0–46.2) 9.0 (1.3–62.8) 0.028 0.636 (0.51–0.76) a: P value, Mann-Whitney U test. b: ROC ( Receiver Operating Characteristics ) – AUC ( Area Under Curve ) analysis c: Median (range) of gene mRNA levels d: AUC value (95% confidence interval) On hierarchical clustering of the samples, the three-gene expression signature dichotomized the 104 patients into two subgroups (n = 30 and n = 74, respectively) of similar sizes to those of the initial patient population (n = 35 and n = 65, respectively). The "poor prognosis" signature was again associated with shorter relapse-free survival in this independent tumor series (log-rank test, P = 0.029; Figure 1B ) (5-year RFS 59.2% ± 9.1 versus 80.7% ± 4.8; 10-year RFS 51.2% ± 9.50 versus 70.4% ± 6.5). Multivariate analysis based on a Cox proportional hazards model showed that, among the parameters that were significant or near-significant (P < 0.2) in univariate analysis, i.e. SBR grade, lymph-node status, macroscopic tumor size (Table 2 ) and the three-gene expression signature (Figure 1B ), only SBR grade was an independent predictor of RFS (P = 0.00023); the three-gene expression signature only showed a trend towards significance (P = 0.27). Discussion We used real-time quantitative RT-PCR assays to quantify the mRNA expression of 47 genes previously identified as candidate prognostic molecular markers in 100 ERα-positive breast tumor samples. We identified a three-gene expression signature ( BRCA2 , DNMT3B and CCNE1 ) with independent prognostic significance in breast cancer (P = 0.007 by univariate analysis; P = 0.006 by multivariate analysis). This "poor prognosis" signature was then tested on an independent set of 104 ERα-positive breast tumors from a well-defined cohort of postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone. It was found to be significant in univariate analysis (P = 0.029), but not in multivariate analysis (P = 0.27). We have previously published individual data for 18 of these 47 genes, namely ERBB1-4 [ 12 ]; MYC [ 13 ]; TERT [ 14 ]; CCND1 [ 15 ]; CGB , CGA , ERα , ERβ , PR , PS2 [ 16 ]; AR [ 17 ]; DNMT3B [ 18 ], PAI1 , PAI2 and UPA [ 19 ], obtained using the same real-time RT-PCR method but in a heterogeneous series of 130 ERα-positive and ERα-negative breast tumors. Large-scale real-time quantitative RT-PCR is a promising complement and/or alternative to cDNA microarrays for molecular tumor profiling. CDNA microarrays have been used to identify gene expression profiles associated with poor outcome in breast cancer [ 20 - 26 ], but discrepancies have been reported. For example, only 2 of 456 genes identified by Sorlie et al. [ 21 ] was among the 70 genes identified by van de Vijver et al. [ 24 ]. These discrepancies may be due to the clinical, histological and ethnic heterogeneity of breast cancer, but also to the fact that breast tumors consist of many different cell types – not just tumoral epithelial cells, but also additional epithelial cell types, stromal cells, endothelial cells, adipose cells, and infiltrating lymphocytes. Real-time RT-PCR requires smaller starting amounts of total RNA (about 1–2 ng per target gene) than do cDNA microarrays, making it more suitable for analyzing small tumor samples, cytopuncture specimens and microdissected samples. Real-time RT-PCR also has a linear dynamic range of at least four orders of magnitude, meaning that samples do not need to contain equal starting amounts of RNA. Real-time RT-PCR is also more suitable than cDNA microarrays for analyzing weak variations in gene expression and weakly expressed genes (e.g. TERT as in the present study), and for distinguishing among closely related family member genes or alternatively spliced specific transcripts (e.g. the gene cluster p14/ARF , p16/CDKN2A and p15/CDKN2B as in the present study). Finally, real-time quantitative RT-PCR assay is a reference in terms of its performance, accuracy, sensitivity and throughput for nucleic acid quantification, and is more appropriate for routine use in clinical laboratories, being simple, rapid and yielding good inter-laboratory agreement and statistical confidence values. In this study, we chose to include well known genes involved in breast carcinogenesis reported in the literature and representing a broad range of cellular functions, such as cell cycle control, cell-cell interactions, signal transduction pathways, apoptosis and angiogenesis (Table 3 ). Many important genes were not studied, but our results nevertheless demonstrate the usefulness of real time RT-PCR by identifying a potentially useful gene expression signature with prognostic significance. The comparison of median target gene mRNA levels between patients who did and did not relapse provided two interesting results: (a) ERBB2 mRNA levels were very similar between the two subgroups, with ROC-AUC values close to 0.5 (ROC-AUC, 0.573), confirming that the ERBB2 mRNA expression level is not a major prognostic factor in breast cancer; (b) ESR1/ERα mRNA levels were not different between the two subgroups (ROC-AUC, 0.530), suggesting that the ESR1/ERα mRNA expression level in ERα-positive tumors is not predictive of outcome. The three-gene expression signature predictive of subsequent relapse status comprised genes involved in cell cycle control ( CCNE1 ), DNA methylation ( DNMT3B ) and DNA damage repair ( BRCA2 ). This gene expression signature is an interesting candidate for routine clinical use, especially as the three genes encode well-characterized proteins for which specific antibodies are already commercially available. Furthermore, the three protein products are amenable to pharmacological control. CCNE1 codes for cyclin E, a protein involved in regulating the early G1 to late G1 phase "restriction point traversal", an irreversible commitment to undergo one cell division [ 27 ]. We found that high CCNE1 mRNA levels were associated with poor outcome, confirming published data suggesting that cyclin E upregulation may be a major prognostic marker in breast cancer [ 28 - 31 ]. BRCA2 codes for a ubiquitously expressed tumor suppressor protein involved in processes fundamental to all cells, including DNA repair, DNA recombination and cell cycle checkpoint control [ 32 ]. We found that high BRCA2 mRNA levels were associated with poor outcome and correlated positively and strongly with cell proliferation. By hierarchical clustering analysis of the 47 genes, we identified BRCA2 as the leading gene in a cluster of proliferation genes also including TERT , BRCA1 , HMMR/RHAMM and MKI67 (data not shown). We also observed a strong positive link between BRCA2 and MKI67 , which encodes the proliferation-related Ki-67 antigen (Spearman rank correlation test: r=+0.670, P < 10 -7 ). The observed strong associations between BRCA2 , HMMR/RHAMM and MKI67 mRNA expression explain why four- and five-gene expression signatures, comprising HMMR/RHAMM alone or together with MKI67 , showed no additional prognostic value relative to the three-gene signature. Our results for BRCA2 expression ex vivo are in keeping with reports from several authors [ 33 , 34 ] showing that BRCA2 mRNA expression is upregulated in rapidly proliferating cells in vitro . Our results are also in agreement with those of Egawa et al . [ 35 ] showing that high BRCA2 expression carries a poor prognosis in breast cancer. This link between BRCA2 overexpression and poor outcome should be taken into account when evaluating future BRCA2 -based therapeutic approaches to breast cancer. Finally, DNMT3B , the third gene in our expression signature, codes for one of the three functional DNA methyltransferases ( DNMT1 , DNMT3A and DNMT3B ) that catalyze the transfer of methyl groups to the 5-position of cytosine (DNA methylation). We previously showed that, among the three DNA methyltransferases ( DNMT1 , DNMT3A and DNMT3B ), only DNMT3B overexpression is associated with poor outcome in breast cancer [ 18 ]. DNMT3B (like DNMT3A ) is known to be a de novo methylator of CpG sites. Abnormal DNA methylation is thought to be a major early event in the development of tumors characterized by widespread genome hypomethylation leading to chromosome instability and localized DNA hypermethylation; the latter may be important in tumorigenesis by silencing tumor suppressor genes [ 36 ]. Conclusions In conclusion, by studying the expression of 47 genes previously identified as candidate prognostic markers in breast cancer, we identified a three-gene expression signature ( BRCA2 , DNMT3B and CCNE1 ) with prognostic significance. The practical value of this signature remains to be validated in large prospective randomized studies. Abbreviations ERα , estrogen receptor alpha; RT-PCR, reverse transcriptase-polymerase chain reaction. Authors' contributions Real-time RT-PCR have been carried out by ST and IG. IB and RL interpreted the result, performed bioinformatics and statistical analyses.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544833.xml
520812	Evaluation of the prophylactic use of mitomycin-C to inhibit haze formation after photorefractive keratectomy in high myopia: a prospective clinical study	Background To study the effect of prophylactic application of mitomycin-C on haze formation in photorefractive keratectomy (PRK) for high myopia. Methods Fifty-four eyes of 28 myopic patients were enrolled in this prospective study. All eyes were operated by PRK followed by 0.02% mitomycin-C application for two minutes and washed with 20 ml normal saline afterwards. All eyes were examined thoroughly on the first 7 days and one month after surgery; 48 eyes (88.9%) at 3 and 6 months postoperatively. Hanna grading (in the scale of 0 to 4+) was used for assessment of corneal haze. Results The mean spherical equivalent refraction (SE) was -7.08 diopters (D) ± 1.11 (SD) preoperatively. Six months after surgery, 37 eyes (77.1%) achieved an uncorrected visual acuity (UCVA) of 20/20 or better, all eyes had a UCVA of 20/40 or better and 45 (93.7%) eyes had an SE within ± 1.00D. One month postoperatively, 2 eyes (3.7%) had grade 0.5+ of haze, while at 3 and 6 months after surgery no visited eye had haze at all. All eyes had a best corrected visual acuity (BCVA) of 20/40 or better and there were no lost lines in BCVA by 6 months after surgery. In spatial frequencies of 6 and 12 cycles per degree contrast sensitivity had decreased immediately after PRK and it had increased 1.5 lines by the 6 th postoperative month compared to the preoperative data. Conclusions The results show the efficacy of mitomycin-C in preventing corneal haze after treatment of high myopia with PRK. This method- PRK + mitomycin-C – can be considered an alternative treatment for myopic patients whose corneal thicknesses are inadequate for laser in situ keratomileusis (LASIK). However, the results should be confirmed in longer follow-ups.	Background In refractive surgery, patients with -5.00 diopters (D) myopia or more and corneal thicknesses less than 500 μm are not suitable candidates for laser in situ keratomileusis (LASIK) or conventional photorefractive keratectomy (PRK) due to the inadequate corneal thickness and risk of haze.[ 1 - 13 ] There are patients in which LASIK may seem possible, but the low pachymetry reading limits us to a small ablation zone, and as a result some post-LASIK complications such as glare and halos may occur. [ 14 ] If haze following PRK can be prevented in myopic eyes with a spherical equivalent (SE) over -5.00 D, obviously there would be less concern about the corneal thickness, ablation zone, and even the flap induced aberrations following Lasik. [ 15 ] Haze after PRK may result from the corneal wound healing process. In animal models, it has been shown that this process is probably initiated by keratocyte apoptosis and the subsequent over-proliferation of cells. [ 16 ] Collagen type IV alpha 3 is also an important factor in the development of corneal haze after PRK. [ 16 ] Mitomycin-C is an antibiotic with anti-metabolite effects that inhibit the proliferation of keratocytes,[ 17 ] but it has no effect on normal epithelial cells of the cornea. Mitomycin-C 0.02% has been used in the treatment of post-PRK haze. [ 18 ] There are also some reports on the prophylactic use of mitomycin-C to prevent haze following PRK in moderate to high myopia. [ 14 , 19 ] In this prospective non-controlled clinical study we have assessed the prophylactic application of mitomycin-C on regression and haze in PRK performed on patients with high myopia (SE ≥ -5.00 D). Methods Study design and patient selection Fifty-four eyes in 28 patients with a spherical equivalent refraction over -5.00 D were included in this prospective non-controlled clinical study. Since the residual stromal bed thickness under the flap after LASIK would be less than 250 μm, no eye had an appropriate corneal thickness for this procedure. In planning PRK, the post-ablation corneal thickness was calculated to be greater than 350 μm. Exclusion criteria in this study were systemic or ocular disease with the potential to interfere with the healing process of the cornea, such as collagenosis, diabetes, dry-eye syndrome, anterior or posterior uveitis, ectatic diseases such as keratoconus, and also corneal dystrophy or degeneration, glaucoma, retinal diseases, lens opacity, history of severe ocular trauma, and previous ocular surgery. The study was approved by the Research and Ethics Committee of the Noor Vision Correction Center. Before the operation, the nature of the procedures, their results and complications were thoroughly explained to all patients and they were asked to read and sign a formal informed consent in their native language. Procedure All treatments were performed between April and October 2002. All eyes underwent the standard PRK procedure by two surgeons with extensive experiences with PRK. Patients received topical anesthesia without systemic sedation. Pre-incision of the corneal epithelium was made using a microtrephine with an 8 mm diameter and 70 μm deep calibrated blade (Janach, J 2900S). The epithelium was removed mechanically with a hocky knife. Then, the ablation was performed using a Technolas 217-C excimer laser (Bausch and Lomb, CA, USA). In all treatments, the overall ablation diameter was 8.4 to 8.9 mm and consisted of a central optical zone of 5.5 to 6.0 mm respectively by a 2.9 mm transition zone. Considering our previous experiences, eyes receiving mitomycin-C were intentionally under-corrected by 5 % compared with LASIK nomogram. Immediately after laser ablation, a single topical application of mitomycin-C 0.02% (0.2 mg/ml) diluted in balanced salt solution was instilled in each eye with a weck sponge placed over the ablated stroma for 2 minutes. The corneal surface and the entire conjunctiva were then vigorously irrigated with 20 ml cold normal saline to remove the residual mitomycin-C. At the end of the procedure, a bandage contact lens was applied which was removed after three days. After surgery, patients were instructed to take an analgesic (diclofenac sodium) every 8 hours and all eyes received artificial tears and flourometholone eye drop every four hours for 2 weeks, and chloramphenicol eye drop every 6 hours for 3 days. During the next two weeks, all patients were treated with flourometholone and artificial tears every 6 hours. These two eye drops were used every 8 and 12 hours during the second and third postoperative months, respectively, and were then discontinued. All patients were instructed to wear sunglasses in direct sunlight for 3 months. The preoperative visit included uncorrected visual acuity (UCVA), best-corrected visual acuity (BCVA), manifest, subjective, and cycloplegic refractions, slit-lamp exams, applanation tonometry, corneal topography, ultrasonic pachymetry, keratometry, indirect funduscopy, and contrast sensitivity with and without glare. UCVA and BCVA were tested with the Snellen chart. On the first seven days after surgery, all patients were examined with a slit lamp and the area of the epithelial defect was measured with its ruler to identify the time of complete reepithelialization. On the 7 th and 14 th days after surgery we measured the UCVA, BCVA, and the manifest, subjective, and cycloplegic refraction. At the 1 st , 2 nd , and 6 th postoperative months, exams and measurements included UCVA, BCVA, manifest, subjective, and cycloplegic refraction, slit-lamp exams, applanation tonometry, ultrasonic pachymetry, keratometry, topography, and contrast sensitivity with and without glare. For evaluation of haze we used Hanna's grading scale from 0 (no haze) to 4+ (dense white corneal haze). Contrast sensitivity was tested by Vector Vision CSV-1000 (Vector Vision, Dayton, OH) in spatial frequencies of 6 (B) and 12 (C) cycles per degree. [ 20 ] The manufacturer's recommended testing procedures were followed. Absolute values of log contrast sensitivity were obtained for each patient and spatial frequency, and means and standard deviations were calculated. Data were then expressed in the notation of normalized log contrast sensitivity values. [ 21 ] Statistical analysis Repeated measures analysis of variance was used to assess changes over time after surgery. For all statistical tests, the significance level was considered 0.05. Results Of the 28 study group patients, 3 did not show up on the 3 rd and 6 th months visits (48 eyes out of 54 were visited; 88.9%). The mean age was 29.3 years (range, 20 to 45 years). The mean preoperative SE was -7.08 D ± 1.11 (SD) (range, -9.88 to -5.00D). The mean preoperative corneal thickness was 488.6 μm ± 11.9 (SD). At the first, 3 rd , and 6 th month after surgery, the mean corneal thickness was 357 μm ± 30 (SD), 373 μm ± 27 (SD), and 380 μm ± 28 (SD), respectively. Thirty-three eyes (61.1 %) had a preoperative BCVA of 20/20 or better, and 51 eyes (94.4 %) had a BCVA of 20/25 or better. Complete reepithelialization was seen in no eye by the 2 nd postoperative day but in all eyes by the 6 th day. The epithelial defect had resolved in 53.7 % of the eyes by the 3rd day, and in 92.6 % by the 4th postoperative day. Figure 1 shows the refractive results in this study group. At the 3 rd postoperative month, 33 eyes (68.7 %) were within ± 0.5 D of emmetropia (SE) and 43 eyes (89.6 %) within ± 1.00D (SE). At the 6 th postoperative month 39 eyes (81.3 %) were within ± 0.5D of emmetropia and 45 eyes (93.7 %) within ± 1.00D. Figure 2 shows the refractive changes after surgery during these six months. The refraction of patients had already become stable one month after surgery. At the 3 rd postoperative month 32 eyes (66.7 %) had a UCVA of 20/20 or better and 47 (97.7 %) of 20/40 or better. All eyes had a BCVA of 20/40 or better and there were no lost lines in BCVA. On the 6 th month visit, 37 eyes (77.1 %) had a UCVA of 20/20 or better and 48 (100%) of 20/40 or better. All eyes had a UCVA of 20/40 or better. On the first month follow-up visit, 2 eyes (3.7%) had +0.5 haze, but all had 0 haze on the 3 rd and 6 th month visits. Figure 3 shows the changes of contrast sensitivity in spatial frequencies of 6 and 12 cycles per degree (CPD) with and without glare over time. In the spatial frequency of 6 CPD, contrast sensitivity had decreased immediately after PRK and it had increased by the 6 th postoperative month compared to preoperative data while in the spatial frequency of 12 CPD, it had increased over time after surgery (P < 0.001). In our study, no complications such as eccentric ablation, delayed reepithelialization, persistent epithelial defect, or microbial keratitis have been observed. Discussion PRK in high myopia remains a challenge due to its complications of haze and regression reported in previous experiments, and also the success rate of LASIK in these patients. [ 19 ] Yet, LASIK cannot be performed in some patients with an insufficient corneal thickness resulting in an undesirable residual stromal bed, or smaller ablation zones had to be applied to correct the refractive error completely. Such a small ablation zone can lead to visual inconveniences such as impaired night vision when the pupil dilates, halos, blurred vision, and ghost images. [ 15 , 22 ] If PRK plus mitomycin-C safety and predictability can be verified, larger ablation zones can be used, therefore the above complications will practically be avoided. On the other hand, correction of higher refractive errors will be possible. This study was carried out to evaluate the safety and efficacy of PRK along with a 2-minute application of mitomycin-C 0.02% (0.2 mg/ml) on the exposed stromal bed after ablation was performed. Mitomycin-C is an antimetabolite and antibiotic drug. It is mostly used systemically in cancer chemotherapy. It has been used in ophthalmology in cases of intraepithelial neoplasms of the cornea and conjunctiva, ocular pemphygoid, and following surgical treatment of glaucoma and pterygium. Mitomycin-C has cytotoxic effects through inhibiting DNA synthesis. The logic behind using mitomycin-C is that the topical application of the drug on the cornea can inhibit subepithelial fibrosis through preventing the proliferation of stromal keratocytes, while the main causes of regression and haze are overacitvity and proliferation of stromal keratocytes following laser ablation. [ 19 ] The effects of mitomycin-C 0.02% in preventing haze has been shown by Talamo et al [ 22 ], and Xu et al [ 23 ] in experimental models. In a study by Majmudar et al, it was concluded that the application of mitomycin-C can remove haze following PRK and radial keratectomy (RK). [ 18 ] The usefulness of PRK with mitomycin-C (0.2 mg/ml) for preventing haze in high myopia was reported by Carones et al. [ 19 ] In this study we used the same concentration of 0.2 mg/ml to perform PRK + mitomycin-C in high myopia; considering the different climate and races in this Middle Eastern region. It seems that the problems of haze and regression after PRK are more prominent in people of this region. [ 24 ] In addition, the inclusion criteria aimed at patients who were at a relatively high risk of haze formation after PRK. Among factors determining the ablation depth, an important predictor of haze, correction and ablation zone size are more important. A 6.0 mm optical zone and a minimum of 5.00 diopters of correction ethically and practically limited our study to those patients who were at a high risk of developing haze with PRK. No immediate toxic effects such as conjunctival chemosis or any delay or irregularity in re-epithelialization were seen. In only one case, after primary reepithelialization a large epithelial defect was seen on the fourth day (one day after complete epithelialization and contact lens removal), which healed in three days by applying a bandage contact lens. No complications such as corneal edema, melting, and perforation were observed in follow-up visits up to six months. Therefore, the topical application of mitomycin-C of the aforementioned concentration and duration was safe in our series. Yet, a longer follow-up is required to assess the long-term complications of mitomycin-C, and its careful application is recommended before long-term complications can be ruled out. According to our previous experiences, we planned a 5% under-correction compared to our usual nomogram for LASIK. As a result of this modification, patients were close to emmetropia with a low variability on their 6 th month visit. In the report by Carones, patients had a hyperopic shift of +0.5D by the sixth month; this is while our patients had a myopic shift of 0.43D. The UCVA at six months after surgery was 20/40 or better in 100% and 20/20 or better in 77.1% of patients. The refractive and visual results of this study are much better compared to reports concerning the same amount of correction with LASIK, [ 25 , 26 ] PRK, or LASEK [ 27 , 28 ]. Considering the fact that haze influences BCVA, patients' BCVA is of great importance. In this study, no eyes lost any lines of BCVA by six months after surgery compared to the preoperative BCVA. None of the eyes had any grade of haze 3 or 6 months after surgery, which shows the efficacy of prophylactic application of mitomycin-C in preventing haze following PRK for patients with myopia over -5.00D. In addition, an average postoperative improvement in contrast sensitivity of 1.5 lines points to the safety and efficacy of the procedure in the quality of vision. In conclusion, using mitomycin-C in PRK for myopia greater than -5.00D seems safe and effective, and can reduce haze formation after surgery; therefore it can be considered a suitable alternative for patients with myopia greater than -5.00 D whose corneas lack an appropriate thickness to perform LASIK with a desirable optical zone. With this method, vision can be corrected with a better quality of vision regarding contrast sensitivity. Competing interests None declared. Authors' contributions HH, SMRT, AF and AK participated in the design of the study and the preparation of the manuscript. HH carried out all operations. SMRT visited patients in the follow up visits. AF and AK participated in the statistical analysis of the study. All authors have read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC520812.xml
538250	The structurally constrained protein evolution model accounts for sequence patterns of the LβH superfamily	Background Structure conservation constrains evolutionary sequence divergence, resulting in observable sequence patterns. Most current models of protein evolution do not take structure into account explicitly, being unsuitable for investigating the effects of structure conservation on sequence divergence. To this end, we recently developed the Structurally Constrained Protein Evolution (SCPE) model. The model starts with the coding sequence of a protein with known three-dimensional structure. At each evolutionary time-step of an SCPE simulation, a trial sequence is generated by introducing a random point mutation in the current coding DNA sequence. Then, a "score" for the trial sequence is calculated and the mutation is accepted only if its score is under a given cutoff, λ . The SCPE score measures the distance between the trial sequence and a given reference sequence, given the structure. In our first brief report we used a "global score", in which the same reference sequence, the ancestral one, was used at each evolutionary step. Here, we introduce a new scoring function, the "local score", in which the sequence accepted at the previous evolutionary time-step is used as the reference. We assess the model on the UDP-N-acetylglucosamine acyltransferase (LPXA) family, as in our previous report, and we extend this study to all other members of the left-handed parallel beta helix fold (LβH) superfamily whose structure has been determined. Results We studied site-dependent entropies, amino acid probability distributions, and substitution matrices predicted by SCPE and compared with experimental data for several members of the LβH superfamily. We also evaluated structure conservation during simulations. Overall, SCPE outperforms JTT in the description of sequence patterns observed in structurally constrained sites. Maximum Likelihood calculations show that the local-score and global-score SCPE substitution matrices obtained for LPXA outperform the JTT model for the LPXA family and for the structurally constrained sites of class i of other members within the LβH superfamily. Conclusion We extended the SCPE model by introducing a new scoring function, the local score. We performed a thorough assessment of the SCPE model on the LPXA family and extended it to all other members of known structure of the LβH superfamily.	Background Protein structure is more conserved than protein sequence during molecular evolution [ 1 - 3 ]. Remote homologous proteins constitute an extreme example of sequence divergence where proteins with similar function and no apparent sequence similarity present almost the same fold [ 4 ]. However, protein sequences are far from being random. Rather, they are selected through evolution in such a way that functional constraints modulate sequence variability. Usually, only a few residues are directly related to the protein function. However, these residues must maintain adequate spatial relationships for the protein to remain functional, so that the whole 3D structure is conserved. In turn, structure conservation constrains sequence variability in such a way that residue substitution does not disturb the overall 3D structure of the protein. This results in emergent non-random sequence patterns. The restrictions imposed by the environment of a given protein site onto its pattern of amino acid substitutions have been largely discussed [ 2 , 5 - 9 ]. Briefly, highly constrained positions are more conserved. Furthermore, each site has a biased composition related to its structural environment. Models of protein evolution that take this into account outperform other, simpler, models [ 10 - 13 ]. Recently, a number of models of protein evolution have been developed that take explicit account of protein structure, stability, and/or foldability [ 14 - 22 ]. Even though such models have not been used yet for phylogenetic inference purposes, they are useful to gain insight into the detailed mechanism of protein evolution. Noteworthy, some of these models have been able to reproduce quantitatively observed amino acid substitution patterns [ 12 , 14 , 23 ]. To study how protein structure conservation modulates sequence divergence, we recently developed the Structurally Constrained Protein Evolution (SCPE) model [ 14 ]. The starting point of an SCPE simulation is the coding-sequence of a protein of known three-dimensional structure, which we shall call the "ancestral sequence". At each evolutionary time-step, a new "trial sequence" is generated by random mutation, at DNA level, of the "current sequence" (accepted at the previous time-step). Then, the trial DNA is translated using the universal genetic code and a "score" that estimates the protein structure perturbation introduced by the mutation is evaluated. The trial sequence is accepted, becoming the new current sequence, only if its score is below a certain "cut-off", λ , that measures the amount of structural perturbation allowed by natural selection. In this way, for λ = 0 only synonymous mutations are accepted, whereas for λ ~ ∞ all mutations are accepted. The procedure is repeated until a desired number of mutations are reached. In the present work the DNA is mutated using the Jukes-Cantor model, so that each nucleotide substitution occurs with the same probability. The model depends on one parameter, the cut-off λ , that must be fit by comparison to actual sequence data. Different properties could be used to fit the cut-off. As we will show below, the model is quite robust with respect to the property used. Therefore, we have used the simplest way, which is to fit λ such that the acceptance rate, ω , inferred for actual sequences is reproduced. The acceptance rate is the probability that an amino acid mutation is accepted. Thus, it can be estimated by the ratio between the number of amino acid substitutions (accepted mutations) and the total number of trial amino acid mutations. The acceptance rate has been extensively used to characterize the strength of the selective pressure under which proteins evolve [ 24 - 27 ]. If all mutations were neutral they would be accepted and ω would be 1. In general the ω values are usually below 0.5 due to the deleterious effects of most amino acid mutations [ 28 ]. In proteins under very strong selective pressure ω can take values very close to zero. One of the main factors determining the quality of the SCPE model is the scoring function. Given the structure of the ancestral protein, which we assume constant throughout the simulation, the score of a given trial sequence is defined as the RMSD between the mean-field energy profile of the trial sequence and that of a reference sequence. In our previous work, the same reference sequence, the ancestral one, was used for each time-step. Therefore, the score of each trial sequence was a measure of the dissimilarity between the trial sequence and the ancestral sequence, given the structure. Such a score depends only on the trial sequence and the ancestral sequence, but not on the particular sequence mutated to obtain the trial. Hence, it does not depend on the precise evolutionary path between the ancestral and the trial. Therefore, this will be called from now on "global score". Even though the global score has been proved to be very good at reproducing the sequence patterns of a test case, it also shows some problems. Mainly, at the beginning of a simulation most mutations fall below the optimum cut-off. This results in too high values of the acceptance rate. Only after about 5% of the sites have been substituted, the cut-off is purifying enough to reproduce the acceptance rate inferred for the actual family. From a more qualitative point of view, since at the beginning of global-score simulations almost all mutations are accepted, erroneous amino acids, which are not found in the natural sequences of the family studied, can be introduced with relatively high probability during the first few steps of a global-score simulation. We shall see below that these are unwanted artefacts of the global-score SCPE simulations. To tackle the problems described in the previous paragraph, in this paper we introduce a "local score", in which the reference sequence for a given trial is that accepted in the previous evolutionary time step, the current sequence, rather than the ancestral one. Thus, the local score measures the mutational perturbation introduced in a given time-step, rather than the global difference between the trial and ancestral sequences. The new approach is compared with the previous one on the same test system studied before: UDP-N-acetylglucosamine acyltransferase (LPXA) from Escherichia coli . A portion of this protein presents a left-handed parallel beta helix (LβH), a fold generally associated with transferase activity and broadly distributed in different taxons [ 29 - 31 ] (see Figure 1a ). All the LβH proteins contain a hexapeptide-repeat motif which is closely related with the topology of the fold (Figure 1b ). This superfamily is characterized by the high conservation of the fold that contrasts with an elevated sequence and functional divergence. Figure 1 (a) Structure of the UDP-N-acetylglucosamine acyltransferase (LPXA). This protein forms a Left-handed parallel β Helix (LβH). (b) Detail of one coil of the helix. Each coil is formed by three hexapeptides (shown in different colours). Note that hexapeptide positions i and i+4 point towards the inside of the prism whereas the other positions point outwards. We shall show below that when the local score is used, the acceptance rate averaged over independent runs does not depend on the amount of divergence from the ancestral sequence. Furthermore, no erroneous amino acids are accepted during the simulations. Thus, these artefacts of the global-score simulations are absent when the new scheme is used. To further compare both schemes, other properties were analysed. Specifically, we evaluated and compared structure conservation, entropy profiles, amino acid distributions, and substitution matrices. We show that SCPE simulations that use the LPXA from E. coli as ancestral sequence can be used to estimate site-dependent amino acid substitution matrices [ 32 , 33 ] which outperform the usually used JTT model [ 34 ]. Moreover, we consider the applicability of the SCPE substitution matrices obtained from LPXA simulations to other protein families which adopt the LβH fold. Results and discussion Acceptance rates In Figure 2 we show the number of nonsynonymous substitutions versus the number of nonsynonymous mutations averaged over several independent simulations. Note that nonsynonymous substitutions (mutations) at DNA level are amino acid substitutions (mutations) at protein level. The slope of each plot is the acceptance rate ω . Figure 2 shows that for the global-score case, ω decreases from ω = 1 when the simulation begins to a constant asymptotic value ω < 1 for longer times. In an actual case, such behaviour could be due to a sequence that for some reason is particularly robust with respect to mutations. In the present case, however, this is an unintended artefact of our model. It happens because the global score of the mutations introduced in the first steps of a simulation lie below the global cut-off, no matter how nonconservative the mutation is. Thus, at the beginning of a global-score simulation almost all amino acid mutations are accepted, leading to an acceptance rate ω = 1. Furthermore, clearly wrong amino acids, that will irreversibly upset the structure, can be introduced. In contrast, the local-score simulations display a constant average ω , which we think is more consistent with a neutral model, such as SCPE, with constant selection pressure λ . We should mention that despite the constancy of the average ω , the acceptance rate ω of a single simulated run changes from sequence to sequence. This is expected, since any substitution at a given site changes the scores of the sites that are in contact with it in the 3D structure. This could account for features such as overdispersion of the molecular clock and rate-shifts in substitution rates (heterotachy). Figure 2 Number of nonsynonymous (amino acid) substitutions observed as a function of the number of nonsynonymous (amino acid) mutation trials for local-score and global-score SCPE simulations. Results are averaged over 300 independent runs. Note that global-score simulations present a definite change in slope (acceptance rate ω ) between the first steps of the simulations and longer times. In contrast, local-score simulations present a constant slope (acceptance rate ω ). Determination of optimal λ As discussed in Methods, we have chosen to determine the optimum value of the SCPE model parameter λ so that the acceptance rate of the simulations matches that inferred from actual sequences. For the SCPE simulations, the value of ω is easily estimated by just counting the number of amino acid substitutions accepted throughout the simulations and dividing by the number of trial amino acid mutations. For the reference alignment, however, one has to estimate ω using some inference method. These methods tend to overestimate the actual ω . This can be seen in Figure 3 , where we show two different ω inferences for a set of SCPE simulations as a function of the cut-off λ , together with the value calculated by counting the proportion of accepted mutations (see Methods). The inferences were made with the module yn00 of PAML [ 35 ] as explained in Methods. It is worthwhile to note that the inferred ω departs from the calculated ω as λ increases. This behaviour is expected since, for a given number of mutations, for larger λ there are more accepted nonsynonymous substitutions, which results in loss of sequence signal. Figure 3 Inferred and calculated acceptance rates of data sets simulated with SCPE. (a) local-score simulations. (b) global-score simulations. yn00 and yn00+w+f are two different methods to infer acceptance rates included in PAML (see Methods). The average acceptance rate inferred in the LPXA reference alignment (obtained with yn00+w+f) is 0.2246 ± 0.11. Using this value in (a) and (b) the optimal local and global λ obtained are 1.10 and 7.00, respectively. In (c) we plot the ω inferred using yn00+w+f versus the calculated value for SCPE runs from (a) and (b). The value inferred for the observed LPXA family is shown as a dotted line. Using this value the optimal ω for local score is 0.15(0.12–0.27) and for global score is 0.19(0.12–0.26). Using the method yn00+w+f, which best estimates the calculated ω , we obtained the ω of the reference alignment of 25 sequences homologous to the UDP-N-acetylglucosamine acyltransferase from Escherichia coli (LPXA reference alignment). The average ω for this alignment is 0.22. Using this value in Figure 3a and 3b the optimal values of λ obtained are 1.10 and 7.00 for local and global score, respectively. We note here that the optimal λ values for local and global score are very different. Thus, for the sake of comparison, we take advantage of the one-to-one relationship between λ and ω , shown in Figures 3a and 3b , and use the calculated acceptance rate ω instead of λ as model parameter. In Figure 3c we plot the inferred ω versus the calculated ω for local-score and global-score simulations. Using this plot and the inferred ω value for the LPXA reference alignment, ω = 0.22 ± 0.11 (0.11 is the standard deviation of ω ), we calculate an optimal ω of 0.15 (0.12–0.27) for local score and 0.19 (0.12–0.26) for global score. Assessment of structure conservation It is important to assess if the SCPE models are able to preserve protein structure. To this end we used THREADER 3 to analyze the percentage of sequences that recognize the correct structure using different models. Results are shown in Table 1 . Clearly, JTT is unable to conserve structure even for relatively low amounts of divergence: at Ka = 0.28 only 20% of sequences obtained from JTT simulations recognize the correct structure. In contrast, a significant proportion of sequences simulated with SCPE recognize the correct structure even after long simulations of 1.7 substitutions per site: 62% for local-score SCPE and 39% for global-score SCPE. Table 1 Evaluation of structure conservation. The table shows the percentage of output sequences that recognize correctly the LβH fold for local-score SCPE, global-score SCPE, and JTT for two different amounts of amino acid substitutions per site (Ka). Amount of Divergence Model Ka = 0.28 Ka = 1.7 Local-score SCPE λ = 1.10, ω = 0.15 87% 62% Local-score SCPE λ = 8.00, ω = 0.92 19% 4% Global-score SCPE λ = 7.00, ω = 0.19 68% 39% Global-score SCPE λ = 90.00, ω = 0.95 8% 0% JTT 20% 0% When both SCPE schemes are compared, Table 1 shows that local-score simulations perform better than global-score ones. This result is counterintuitive, because one might expect, a priori , that in the long term the global-score would be better at conserving structure than the local-score, since in the later case the reference sequence is reset at each step so that it would be easier to lose memory of the ancestral protein. One of the reasons of the global-score SCPE being worse at conserving structure could be the erroneous amino acid substitutions introduced at the beginning of the simulations (see above). To gain more insight into this issue, further work involving much longer simulations would be needed. However, for long enough evolutionary time it is not longer reasonable to assume that structure remains constant. In this limit, any model based on assuming structural conservation will break down. Entropy profiles To evaluate the capacity of the SCPE model to reproduce the sequence patterns found in the LPXA family, the variability of each site was analysed. The different protein positions were accumulated into 6 structural classes. For each class, we calculated the entropies corresponding to the equilibrium distributions of SCPE models. These entropies represent the average structural constraints of each structural class and do not depend on simulation time. SCPE entropy profiles are compared with those obtained from the reference alignment of the LPXA family. One could argue that these are not only determined by structure, but also contain historical information. However, since we are accumulating over several sites of the same class, which would have independent evolutionary histories, we expect such information to be somewhat averaged out. For the sake of comparison we also calculated the entropy profiles of JTT simulations of 0.28 amino acid substitutions per site (see Methods). The resulting entropy profiles are shown in Figure 4 . It can be seen from this figure that both, the local-score and the global-score schemes reproduce very well the variability pattern of the LPXA family. Also, in Figure 4 we show that simulations performed using the JTT model produce less accurate results, especially for the most conserved (low entropy) structural classes i and i+4 . Figure 4 Entropy profiles. Each structural class corresponds to a particular position in the hexapeptide motif found in the LβH proteins. Structural classes i and i+4 are the most conserved while the other classes present a more variable composition. SCPE profiles correspond to equilibrium amino acid distributions (see Methods). The SCPE parameters were fit to the minimum of the entropy error (see Figure 5). The profile obtained from JTT simulations of 0.28 substitutions per site is shown for comparison. To further study the effect of varying the model parameter, we calculated an "error" which quantifies the difference between simulated and observed entropy profiles (see Methods). In Figure 5 this error as a function of ω is shown. Comparing Figures 5 and 3 , we see that the ω for which the entropy error is minimum is consistent with the value at the optimum cut-off for both the local and the global score schemes. Figure 5 Error in entropy profiles between observed and equilibrium SCPE amino acid probability distributions versus calculated acceptance rate. Results for local-score and global-score SCPE simulations are shown, together with those obtained from JTT simulations of 0.28 amino acid substitutions per site. Probability distributions Although entropy is commonly used to evaluate sequence conservation in an alignment [ 36 - 38 ] and to compare simulated data with natural sequences [ 39 , 40 ], it is not enough for a thorough assessment of the sequence pattern. An entropy value of 0 at a given site, for example, means that there is only one amino acid, but it could be any one out of twenty. Thus, to perform a more complete evaluation of the SCPE model, we looked into the amino acid probability distributions. To this end, we calculated a similarity score between the asymptotic SCPE distributions and those obtained from observed sequences. We used the similarity score used by Yona and Levitt to perform sequence profile-profile comparisons [ 41 ]. In Figure 6 we show the similarity score between observed and SCPE equilibrium amino acid distributions as a function of the calculated acceptance rate ω . We also show results for a simulation performed using the JTT model [ 34 ] of evolution. Overall, it can be seen that the local-score SCPE performs somewhat better than the global-score SCPE, and that both SCPE models clearly outperform JTT for a significant range of parameter ω around the optimum value. Figure 6 Similarity score between observed and equilibrium SCPE amino acid probability distributions versus calculated acceptance rate. Results for local-score and global-score SCPE simulations are shown, together with those obtained from JTT simulations of 0.28 amino acid substitutions per site. For ω = 0, the distribution is that obtained from the ancestral sequence, LPXA of Ecoli by grouping sites of the same class, since in this case no substitution is accepted and therefore it is impossible to obtain the SCPE substitution matrices. For SCPE we used equilibrium distributions, which do not depend on time. JTT results become worse for longer times (Ka>0.28). A more detailed analysis shows that the maximum of the local-score plot corresponds to a ω = 0.12, that is in good agreement with the optimum cut-off determined from the acceptance rates, as explained previously. In contrast, for the global-score case the cut-off at the maximum of the similarity score plot is significantly below the optimum ω value previously obtained. This difference would be due to the wrong behaviour of the global-score scheme for small amounts of divergence (see Figure 2 ), which will affect the SCPE substitution pattern and, therefore, the amino acid probability distributions. The same behaviour, though less marked, is found in the plots of Figure 5 . Finally, it is interesting to note that the similarity score for ω = 0 is much better than JTT. Since ω = 0 corresponds to a simulation where no nonsynonymous substitutions are accepted, this is the score obtained using just the initial sequence. Memory of this sequence might favour the good agreement observed for SCPE. However, it is noteworthy that the actual agreement increases for ω > 0, showing that the good fit is not due exclusively to a memory effect. The substitution matrix assessment described in the next section should be less sensitive to memory effects. Substitution matrices Even though it has long been recognized that substitution patterns are site-specific and depend on protein family, it is in general very difficult to estimate site-specific and family-specific substitution matrices due to a lack-of-data problem. As we reported previously, a possible strategy to overcome this obstacle is to obtain site-specific substitution matrices from SCPE simulations [ 12 ]. To further evaluate how the SCPE model is able to reproduce the substitution pattern of the LPXA family, a maximum likelihood analysis was used. SCPE runs were used to obtain a substitution matrix Q c for each structural class. Then, these matrices were used to calculate the maximum likelihood of the LPXA reference alignment using a given topology (see Methods). In Figure 7a we show the likelihood vs. ω plots obtained using local-score and global-score SCPE substitution matrices. The global-score likelihood peaks near ω = 0.18 in good agreement with the previous determinations, showing that it reproduces quite well the amino acid substitution patterns found in real sequences. The best ω of the local-score SCPE likelihood (see Figure 7a ) corresponds to ω = 0.4, larger than that determined previously (Figures 3 , 5 , and 6 ). To understand this behaviour, we analysed the log likelihood components for each structural class, which are shown in Figure 7b . It is seen from this figure, that the local-score maximum likelihood peaks near ω = 0.4 mainly because of the contributions of structural classes i+1 , i+2 , i+3 , and i+5 , which, being the least structurally constrained sites, are not expected to be very well reproduced by SCPE. In contrast, for those sites that point towards the inside of the LβH helix, which are the ones the model should best describe (conserved classes i and i+4 ) the maximum likelihood peaks near ω = 0.2, in better agreement with Figures 3 , 5 , and 6 . In the global-score case, from Figure 7b , the maximum likelihood plots for different classes behave more evenly: for all classes, the maximum likelihood peaks near ω = 0.15. Figure 7 Maximum likelihood as a function of calculated acceptance rate. (a) Likelihood obtained using local-score and global-score SCPE substitution matrices as a function of ω . We also show the likelihood obtained using JTT. (b) Likelihood for the six structural classes using local-score and global-score SCPE matrices. Figure 7a shows that for LPXA, local-score simulations lead to better substitution matrices than global-score ones. Inspection of Figure 7b reveals that this is mainly due to the local-score SCPE giving better results for sites i+4 and, to a lesser degree, i+2. Figure 7a also reveals that both, local and global, SCPE models outperform JTT (dotted line of Figure 7a ) for almost the whole ω range studied. This is due to the fact that site-specific amino acid substitution patterns, especially for constrained structural classes i and i+4 , are not well described by general models such as JTT. Other LβH families As a further example of the applicability of the SCPE model, we considered other families of the LβH superfamily (see Table 2 ). We used the local-score and global-score schemes with the optimum cutoffs estimated using acceptance rates, as explained previously, to obtain site-dependent probability distributions and substitution matrices for the six different structural classes. Table 2 LβH superfamily members studied. Gene name or synonym Function PDB ID Number of sequences aligned LPXA UDP-N-acetylglucosamine acyltransferase 1lxa 25 SATA Streptogramin A Acetyltransferase 1kk6 48 LACA Galactoside O-Acetyltransferase 1kru 43 CAT Xenobiotic Acetyltransferase 1xat 39 DAPD Tetrahydrodipicolinate-N-Succinlytransferase 1tdt 50 CAM Carbonic Anhydrase 1qre 26 GLMU N-Acetylglucosamine-1-Phosphate Uridyltransferase 1g97 50 In Figure 8 we show the probability distributions of the LβH families considered (Figure 8a ) and the equilibrium distributions obtained using the local-score and global-score SCPE models (Figure 8b ). It can be seen that both SCPE schemes perform quite well in reproducing the sequence pattern of our test system. Figure 8 Amino acid frequency distributions for the hexapeptide sites. (a) 7 LβH families of Table 2. (b) Local-score (grey) and global-score (black) SCPE equilibrium distributions (see Methods). To test the substitution matrices, we performed maximum likelihood calculations on each family of Table 2 . Since the models compared have the same number of parameters, they can be compared using Maximum Likelihood (ML) values obtained using a reasonable phylogenetic tree topology [ 42 , 43 ] (see Methods). In Table 3 we show the ML values per site for local-score SCPE, global-score SCPE, and JTT, applied to different sets of sites. Better models have larger ML values. Table 3 Comparison of models on 7 families of the LβH superfamily. Logarithm of the Maximum Likelihood per site obtained with different models for the families studied. Better models lead to larger ML values. The three numbers reported for each case correspond to structural classes i and i+4 , considered separately, and to the average over the six structural classes. Family Local score Global score JTT model LPXA -19.9 -20.0 -23.7 -21.7 -22.7 -24.9 -28.0 -28.1 -29.3 SATA -16.2 -16.1 -18.7 -20.1 -19.2 -20.2 -25.2 -24.7 -25.9 LACA -35.7 -34.2 -36.3 -36.4 -35.0 -35.9 -38.0 -37.0 -37.4 CAT -13.8 -13.7 -16.4 -15.8 -15.9 -17.4 -19.3 -19.0 -19.8 DAPD -17.0 -17.1 -19.6 -22.0 -23.8 -23.2 -18.7 -20.0 -19.4 CAM -13.9 -14.1 -15.9 -20.3 -18.5 -18.6 -23.1 -21.2 -20.4 GLMU -38.1 -38.0 -39.9 -39.0 -38.1 -38.1 -49.3 -47.8 -47.9 For LPXA, SCPE (both local and global) are clearly better than JTT for all sites considered. CAT and SATA behave similarly, though the advantage of SCPE over JTT is less marked. For other families, SCPE (local and global) is better than JTT for class i sites. For other structural classes there is no definite advantage of SCPE over JTT. When comparing local-score SCPE with global-score SCPE one finds no definite advantage of either one over the other. For sites i , where the more meaningful results are expected, local and global give very similar results for all families except for LACA where global is better than local. Conclusion We presented in full detail the Structurally Constrained Protein Evolution Model (SCPE), developed recently. We improved on our previous model by introducing a new scoring function. Our previous work was based on a "global" score, which measures how a trial sequence differs from the ancestral sequence in its ability to fit a reference structure assumed constant. In contrast, the "local" score measures the perturbation introduced by a given mutation with respect to the previously accepted sequence, rather than the ancestral one. Both schemes, global and local, were compared in their ability to match the substitution patterns of the protein family LPXA. We performed a thorough assessment comparing structure conservation, entropy profiles, amino acid distributions, and substitution matrices. LβH proteins were found to be particularly suited for such a detailed characterization of the sequence pattern, because of the fact that most of their sites belong to one of only six different structural classes. Furthermore, these properties were studied as a function of the single parameter of the model: a cutoff that measures selection pressure against structural divergence. Finally, we applied the model to all other members of the LβH superfamily whose structure is known, extending previous studies performed only on the LPXA family. In general, we found that the local-score SCPE behaves either similarly or better than the global-score scheme, depending on the property considered. Furthermore, for LPXA, and for sites of the structurally constrained class i of all other families studied, both SCPE models clearly outperform the widely used JTT model, showing the power of the SCPE model to account for substitution patterns conditioned by structural constraints. Currently, we are using the SCPE model to investigate several issues important in protein evolution, such as overdispersion of the molecular clock, correlation between the evolution of different sites, and heterotachy. Also, we are testing the applicability of the SCPE model to other protein families, in order to assess its generality. Nevertheless, we should mention that since most protein families do not display the regularity of LβH proteins, it is more difficult to perform a detailed quantification of sequence patterns, which makes such tests at the same time more difficult and less demanding than the LβH superfamily. Methods Test system The LPXA family belongs to a large and diverse group of proteins [ 31 ], the LβH (Left-handed parallel β Helix) superfamily. All the sequences of this superfamily contain an imperfect tandem-repetition of a hexapeptide motif [ 29 ]. This motif is typically described by [LIVMA]-X 3 - [ASCVTN]-X. The first position of the hexapeptide is called i , and the following i+1 , i+2 , up to i+5 . The sequence forms a left-handed parallel β helix, forming an equilateral triangular prism [ 44 ] (Figure 1a ). Each coil of the helix is formed by three hexapeptides. Equivalent positions of different hexapeptides fall into similar structural environments. Residues at positions i and i+4 , for example, point towards the inside of the β helix (Figure 1b ). Thus, each site of the hexapeptide pattern corresponds to a different structural class. In this study we did not analyse sites that are at loop regions. Also, the first and last coils of the β helix of LPXA were not considered, since the structural environments of sites in these coils are not exactly the same as those of the other coils. Although all the LβH members have a homo-trimeric active form, we only use the monomer form in this study. We also analyse other LβH families, which are summarized, with a brief description, in Table 2 . SCPE score The first step in the calculation of the SCPE score is the calculation of a profile of mean energies per position. In the present case we used the Cβ-Cβ potential of the program PROSA II [ 45 ]. The original coordinates of the ancestral sequence were modified in order to provide with Cβ coordinates to those residues without them. Thus, all the GLY residues were substituted for ALA residues and an adequate rotamer was chosen using the program SCWRL [ 46 ]. Later, the substituted ALA residues were converted back to the original GLY, keeping the Cβ coordinate of ALA to use when a GLY mutates to a residue with Cβ. Once the energy per position is obtained the score is calculated using: where N is the length of the protein sequence, E mut (p) is the mean-field energy of position p in the trial (mutated) sequence and E ref (p) is the corresponding value of the reference sequence. The "global score" is calculated using the ancestral sequence as reference. The "local score" is calculated using the sequence accepted in the previous step in the simulation (i.e. the sequence that is mutated to obtain the trial). SCPE simulations The ancestral sequence was the UDP-N-acetylglucosamine acyltransferase (LPXA) from Escherichia coli . The coordinates were obtained from the PDB database [ 47 ] (ID code 1lxa). The cutoff range covered was 0–2.00 with a step of 0.1 for local score and 0–20 with a step of 1.00 for global score. For each cutoff value we performed 300 independent simulations, each one of 2500 mutational steps. Sequence analysis Using the LPXA from Escherichia coli as the reference protein, we recovered 25 homologous sequences using sequence similarity searches. This set constitutes the reference LPXA family. For each of the other members of the LβH superfamily for which at least one member has known structure, we used this member's sequence to characterize putative homologous proteins. See Table 2 for details. All the similarity searches were performed using the program BLASTP [ 48 ] at the NCBI server and the sequence alignments were obtained using Clustal X [ 49 ]. Estimation of acceptance rates To assess the optimal selective pressure in our SCPE simulations, we inferred the mean ω value in the homologous LPXA family. Also, we inferred the ω in our SCPE simulations for different cut-offs. All the ω inferences were made using the program yn00 from PAML [ 35 ]. We used options "w", which applies a weighting scheme between codons, and "f", which takes into account the codon frequencies of the data. In the SCPE simulations, we also estimated ω directly by counting: ω is the ratio between the number of amino acid substitutions (accepted mutations) and the total number of amino acid mutation trials. We use "calculated", as opposed to "inferred" to designate the acceptance rates obtained in this way. Estimation of the amount of divergence Some of the comparisons performed depend on the amount of divergence. For these cases, we estimated the average divergence of the LPXA family using the program PAML[ 50 ]. Maximum likelihood distances were estimated using the JTT model with the frequencies estimated from the data and a gamma distribution with 8 categories to estimate the relative rates (JTT+F+Γ). The average time calculated was Ka = 0.28 amino acid substitutions per site. Assessment of structure conservation We evaluated whether sequences produced by evolutionary simulations using SCPE recognize the correct structure using THREADER 3 [ 51 ]. We considered the following schemes: local-score SCPE with λ = 1.10 ( ω = 0.15); local-score SCPE with λ = 8.00 ( ω = 0.92); global-score SCPE with λ = 7.00 ( ω = 0.19); global-score SCPE with λ = 90.00 ( ω = 0.95). To compare, we also ran simulations using JTT. For each model, we performed 50 independent runs of lengths Ka = 0.28 and Ka = 1.7 amino acid substitutions per site. For each sequence, structure recognition using THREADER 3 was performed. The ability of models to conserve structure was measured by the percentage of sequences which recognized correctly (Z-score > 2.7) the LβH fold. Substitution matrices Site-specific replacement matrices are obtained straightforwardly by "counting" substitutions in SCPE simulations. For the test system considered, sites can be classified into c = 1,2,...6 site classes. Then, for each class we set up a matrix of counts: for i ≠ j , is half the number of mutational steps that result in either i → j or j → i amino-acid replacements at site class c , and is the number of mutational steps for which amino acid i remains constant ( i → i replacement). Then, for each class, the matrix of substitution rates, Q c , is obtained using: Given the rate matrices, Q c , the probability matrices are obtained using P c = exp( t Q c ) The vector of amino acid equilibrium frequencies of class c is, then, obtained with Since there are some substitutions that do not occur during the simulations (very low probabilities), we have found it convenient to re-calculate each Q c using a pseudocounts procedure similar to that developed by Tatusov [ 52 ] as follows where and are, respectively, the substitution matrix elements and equilibrium frequencies of a reference model. Here we used JTT [ 34 ] and α = 0.01. Accordingly, equilibrium frequencies were also corrected using Entropies and amino acid distributions To study the sequence variability profile, we calculated the entropy for each structural class using: where is the probability of finding residue i at structural class c . For SCPE, we used the equilibrium probabilities obtained from the substitution matrices, as described in the previous section. For the reference alignment, we grouped all columns of the same structural class together, counted the number of times each amino acid occurred in each class, and obtained the corresponding amino acid frequencies. The difference between the entropy profiles obtained from the SCPE models, , and the profile of the observed reference family, , was quantified by the following "error" function: To assess the similarity between the equilibrium SCPE amino acid distributions and those obtained from the reference alignment, we used the similarity score based on information theory proposed by Yona and Levitt [ 41 ]. The score is calculated by adding together the similarity scores of the six structural classes. JTT distributions and entropies The equilibrium SCPE distributions and their corresponding entropies were compared with JTT distributions and entropies. In contrast to SCPE, the equilibrium JTT distribution does not depend on structural class. Therefore, instead of the equilibrium distributions, we chose to use the distributions and entropies from the alignment of sequences obtained from simulations with the JTT model. To this end, we performed 100 independent simulations using the JTT substitution matrix. The simulation length was set to the average number of substitutions obtained for the LPXA family (Ka = 0.28). We aligned the 100 output sequences, grouped all columns of the same structural class together, counted the number of times each amino acid occurred in each class, and obtained the corresponding amino acid frequencies. Maximum likelihood calculations In order to assess the SCPE substitution patterns, we performed Maximum Likelihood (ML) calculations using the site-dependent SCPE substitution matrices, Q c . The maximum likelihood of a model, Q , given the data, s , for topology, T , is obtained by maximizing the probability L = Pr( s \| T , Q ). For the SCPE model, the reference alignment was partitioned into 6 sub-alignments corresponding to the 6 structural classes. Using these sub-alignments and the corresponding SCPE Q c matrices, we calculated the maximum likelihood using PAML. In all cases a gamma distribution was used to take into consideration the rate heterogeneity among sites of the same class. Similarly, we performed ML calculations using the JTT substitution matrix with gamma distribution of rates (JTT+Γ), for each of the six structural classes. The ML values obtained for each class were added together to obtain the total ML, as was done with the SCPE models. It has been shown that as long as the tree topology is reasonable, model comparison is robust with respect to variations in topology [ 43 ]. In the present case, topologies were obtained using the program FITCH [ 53 ] of PHYLIP 3.57c [ 54 ] with ML distances obtained using JTT with PAML. All the models compared here have the same number of parameters. Therefore, models were compared by comparing ML values. One should note, however, that when models with different number of parameters are compared, one should use a statistic that takes explicit account the number of parameters of each model [ 42 , 43 ]. Authors contributions GP and JE developed the mathematical model. GP implemented the model, run the simulations, performed the analysis and wrote the first draft. JE edited and wrote the revised versions. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC538250.xml
546201	The association of spatial T wave axis deviation with incident coronary events. The ARIC cohort	Background Although current evidence suggests that the spatial T wave axis captures important information about ventricular repolarization abnormalities, there are only a few and discordant epidemiologic studies addressing the ability of the spatial T wave axis to predict coronary heart disease (CHD) occurrence. Methods This prospective study analyzed data from 12,256 middle-aged African American and white men and women, from the Atherosclerosis Risk in Communities Study (ARIC). Following a standardized protocol, resting standard 12-lead, 10-second electrocardiograms were digitized and analyzed with the Marquette GE program. The median follow-up time was 12.1 years; incident coronary heart disease comprised fatal and non-fatal CHD events. Results The incidence rate of CHD was 4.26, 4.18, 4.28 and 5.62 per 1000 person-years respectively, across the spatial T wave axis quartiles. Among women for every 10 degrees increase in the spatial T wave axis deviation, there was an estimated increase in the risk of CHD of 1.16 (95% CI 1.04–1.28). After adjustment for age, height, weight, smoking, hypertension, diabetes, QRS axis and minor T wave abnormalities, this hazard rate ratio for women fell to 1.03 (0.92–1.14). The corresponding crude and adjusted hazard ratios for men were 1.05 (95% CI 0.96–1.15) and 0.95 (0.86–1.04) respectively. Conclusions In conclusion, this prospective, population-based, bi-ethnic study of men and women free of coronary heart disease at baseline shows that spatial T wave axis deviation is not associated with incident coronary events during long-term follow up. It is doubtful that spatial T wave axis deviation would add benefit in the prediction of CHD events above and beyond the current traditional risk factors.	Background Ventricular repolarization abnormalities play an important role in the determination of arrhythmia and sudden cardiac death [ 1 , 2 ] and may reflect subclinical myocardial ischemia changes. The process of repolarization at rest is routinely quantified from standard 12-lead electrocardiogram (ECG), either as time-domain indexes such as the QT interval and its derivations, or as abnormalities of the ST segment or of the T wave. While these indexes have all been reported to be associated to some degree with incident coronary heart disease events, they have some limitations. The widely used QT interval reflects only the temporal aspect of the repolarization, and as currently defined and measured from the 12-lead ECG, QT dispersion has major conceptual and technical limitations [ 3 , 4 ]. Theoretical and experimental studies suggest that ventricular repolarization occurs in a nonlinear and inhomogeneous fashion [ 5 - 10 ]. As a consequence, spatial measures of repolarization that take into account T-wave complexity using the T-wave vector (axis) should be more accurate and useful surface ECG markers of repolarization abnormalities than simple scalar intervals from the ECG, such as the QT interval or QT dispersion [ 11 - 16 ]. Clinical studies have shown that the T wave axis reflects changes associated with autonomic adaptive or maladaptive influences [ 17 ], systemic hypertension [ 18 , 19 ], coronary occlusion [ 20 ], and microalbuminuria in individuals without diabetes mellitus [ 21 ]. Moreover, it has recently been shown that spatial T wave axis deviation has good measurement properties and is repeatable [ 22 ], a finding that supports its use in clinical and epidemiological research. Two prospective population-based reports on T wave axis deviation measured from standard 12-lead ECGs in older populations (the Rotterdam study [ 23 ], and Cardiovascular Health Study (CHS) [ 24 ] suggest that it is an indicator of increased risk of coronary heart disease and total mortality, independent of other cardiovascular risk factors. In the cohort of high-risk, middle-aged men from the Multiple Risk Factor Intervention Trial (MRFIT), baseline spatial T wave axis deviation was not significantly associated with incident coronary events, although the change over time in the spatial T wave axis deviation was reported to be associated with incident events on long-term follow-up [ 25 ]. These studies suggest that spatial T wave axis deviations capture changes in the ventricular repolarization process that are of potential clinical and epidemiologic importance. The aim of this study was to assess whether a single base-line measurement of spatial T wave axis deviation is associated with prospectively ascertained coronary events in a population-based, bi-ethnic cohort of middle-aged men and women, to determine if such an association is independent of other risk factors, and to compare its association with that of other indexes of altered repolarization, such as a prolonged QT interval and minor T wave abnormalities. Methods Between 1987 and 1989, the Atherosclerosis Risk in Communities (ARIC) study examined population-based samples of residents aged 45 to 64 years from 4 communities in North Carolina, Mississippi, Minnesota, and Maryland. From the initial sample of ARIC baseline participants (n = 15,792) we excluded those not having an ECG (n = 205), and because of small numbers, self-identified ethnicity other than white or African American (n = 48). Further, we excluded participants likely to present secondary repolarization abnormalities such as myocardial infarction (self-reported or ECG-diagnosed, n = 790), use of digitalis or anti-arrhytmic drugs (n = 115), or pathologies likely to alter the measurement or interpretation of the spatial T wave axis as identified by the Minnesota code (MC), such as major Q or QS waves (MC 1.1, 1.2), ST depression or elevation (MC 4.1 to 4.4 and MC 9.2), negative T waves (MC 5.1 or 5.2), WPW pattern (MC 6.6), ventricular conduction defects (MC 7.1, 7.2, 7.4, and atrial fibrillation or flutter (MC 8.3), (n = 2093). Participants who had ECG evidence or history of myocardial infarction, coronary bypass, or angioplasty) (n = 162) at baseline were excluded from analysis. Participants whose angina status was positive or unknown by the Rose questionnaire were not excluded because the questionnaire's validity, especially in women, has been questioned [ 26 ]. The final study sample was 12,256 participants. Baseline measurements ECG measurements, processing, and definition of the spatial T wave axis During a fifteen-minute supine rest, trained and certified technicians positioned disposable Ag/AgCl electrodes and recorded a supine, resting 12-lead, 10-second electrocardiogram using a MAC Personal Cardiographer™ (Marquette Electronics, Inc., Jupiter, FL). Detailed procedures used for electrode placement and skin preparations are described in the Operation Manual [ 27 ]. ECGs were digitized at 250 Hz and sent daily via modem to the Epidemiological Cardiology Research (EPICARE) Center, North Carolina. The EPICARE Center, blinded to participant identity processed the ECGs using the most recent version of the Marquette GE program, version 12SL. Quality assurance procedures were designed specifically for ECG acquisition and processing [ 27 ]. These programs included: standardized electrode positions were assured by marking of the skin using a standardized flexible ruler according to a detailed procedure manual. Electrical noise, overall and beat-to-beat electrocardiogram drift were monitored and quality-scored using a five level grading system to assist technicians in identifying unacceptable electrocardiograms and maintaining quality. Technical performance criteria were established and monitored by the EPICARE Center. All technicians were specifically trained and certified. All ECGs were read according to the Minnesota code [ 28 ] without knowledge of clinical or demographic data. All resting 12 lead ECGs coded as having key abnormalities by Minnesota codes including any 1-code, any 1-2 code or 2-2, 5-1 or 2 or any 9-2, 6-4, 7-1-1 or 7-2-1 codes and a 10% random sample of all other ECGs read by computer also were visually coded at the Minnesota ECG Reading Center. Adjudication of discrepancies was performed at the Minnesota Center. The computer-assigned codes are used as study data except where adjudication resulted in a code different from the original computer codes. Spatial T-wave axis was calculated as previously described [ 24 ] from integrated T-wave amplitudes of the XYZ leads. Briefly, the inverse transformation by Dower et al. [ 29 ] was used to derive Frank's XYZ leads, with the polarity of the Z lead inverted in order to generate QRS and T wave patterns with a more familiar waveform in that lead. Thus, the positive direction of the Z axis is in the anterior direction. Spatial T-wave axis was calculated from the scalar product between the T vector and a unit vector in a normal reference direction (x = 1/√3, y = 1/√3, and z = -1/√3, where x, y and z are the unit vector components in the X, Y and Z directions). T axis expresses spatial T vector deviation from the approximate normal direction of the T vector 45° anteriorly in the XZ plane and at 45° elevation from the Y axis. The repeatability of the spatial T wave axis deviation was determined using identical study procedures in a group of apparently healthy volunteers with demographic characteristics similar to the ARIC participants. The repeatability of the spatial T wave axis deviation was high with an intra-class correlation coefficient of 0.87 [ 22 ]. The Cornell voltage index was determined as the sum of R wave amplitude in leads aVL and the S wave amplitude in lead V3 [ 30 ]. The QT interval from the digital 12-lead ECG was determined by the NOVACODE program [ 31 ]. An overall QT interval was calculated from the common QRS onset and T offset for all 12 leads together. To attempt correction for the heart rate dependence, we used several approaches: Bazett's and Fridericia's formulae as well as the QT Index. For comparability with other studies we report the QT interval "corrected "by the Bazett's formula [ 32 ], in spite of its well-known limitations. Covariate measurements The baseline examination was carried out in 1987–1989 and consisted of a home interview of all potential cohort members including items on cardiovascular risk factors, socioeconomic factors and family medical history. After obtaining informed consent, the clinic examination consisted of medical history interview, blood pressure and anthropometric measurements, venipuncture for blood samples and a 12-lead standard electrocardiogram. Anthropometrics were measured with participants wearing scrub suits and no shoes. Height, measured to the nearest centimeter and weight, measured to the nearest pound, were used to calculate body mass index (BMI). Waist-to-hip ratio was defined as the waist girth at the umbilicus (centimeters) divided by the maximum girth of the hips (centimeters). Smoking status was defined as "current smoker" if the person answered "yes" to both of the following questions: "Have you ever smoked cigarettes? and " Do you now smoke cigarettes?". Cholesterol and triglycerides were measured enzymatically. LDL-cholesterol was calculated. Hypertension was defined as having systolic blood pressure values equal or higher than 140 mmHg, or diastolic blood pressure values equal or higher than 90 mmHg or use of blood pressure lowering medication use in the past two weeks [ 27 ]. Ascertainment and classification of incident CHD cases Deaths and hospitalization events were ascertained by annual follow-up calls to the cohort members, review of vital records, and community surveillance of hospitalized and fatal events. CHD death was defined as lacking a probable non-CHD cause, and occurring in the context of a recent myocardial infarction, chest pain within 72 hours of death, or a history of CHD. Events were classified independently by two members of the Mortality and Morbidity Classification Committee and discrepancies were adjudicated by a third member. Descriptions of event ascertainment and classification have been published [ 33 , 34 ]. For the present study, we included CHD events occurring between the ARIC baseline examination and December 31, 2000. The median follow-up time was 12.1 years (maximum of 14.1). We defined CHD incidence as (1) a definite or probable MI, (2) a silent MI between examinations ascertained by ECG, or (3) a definite CHD death. Data analysis We treated spatial T wave axis deviation as a continuous variable. For the purpose of comparison, however we categorized the spatial T wave axis deviation using thresholds employed in previously published reports: normal (<30 degrees), borderline (30 to 45 degrees), and abnormal deviation (>=45 degrees). Cox proportional hazards regression [ 35 ] was used to model time-to-event in the presence of censoring. Time-at-risk (time-to-event or time-to-censoring) was calculated from the date of the baseline examination to the earliest of the following: date of coronary event, date of death, date of last follow-up contact, or December 31, 2000. Hazard ratios with 95%confidence intervals were derived for each variable. When treating spatial T wave axis as a categorical variable, dummy variables were created. Interactions between spatial T wave axis and gender, QRS axis, minor T wave abnormalities and QT interval, were tested as cross-product terms in the multivariable model. Significance of interaction was assessed based on the likelihood ratio test (p < 0.10). Statistical analyses were conducted for each gender to allow for well-known differences in ventricular repolarization by gender, and because of a statistically significant interaction between spatial T axis and gender on CHD events. SAS version 8.1 (SAS Institute, Cary, North Carolina) was used in all analyses. Results Sample characteristics The study population consisted of 12,256 participants, of which 7,143 (58.3%) were women and 3,050 (24.9%) were African American. The mean (SD) age of the study population at baseline was 53.8 (5.7) years. The cohort was followed up for a maximum of 14.1 years [mean (SD) 11.6 (2.3) years]. During this time 653 incident coronary heart disease events occurred. Of the total number of events, 250 coronary heart disease events occurred among women (cumulative incidence 3.5%) and 403 (cumulative incidence 7.9%) among men. The average annual incidence rate was 4.6 per 1000 person-years (95% CI, 4.2–4.9 per 1000 person-years). Correlates of spatial T wave axis deviation The distribution of the spatial T wave axis deviation at baseline was similar for men and women, with a mean (SD) of 23.9 (10.7) degrees in men and 23.2 (11.6) degrees in women. Several traditional cardiovascular risk factors showed graded associations across the spatial T wave axis deviation quartiles, with similar patterns among both men and women (Table 1 ). Participants of African American ethnicity, patients with hypertension and diabetes, and those with higher Cornell voltage presented larger values of spatial T wave axis deviation; lower values for the spatial T wave axis were observed among participants with larger values of the QRS axis, T wave axis in the frontal plane and among smokers. We considered the frontal plane T wave axis for ensuring comparability with other published studies. Other covariates analyzed were anthropometric measures (weight, BMI, waist-to-hip ratio), systolic and diastolic blood pressure, glucose levels, total-, and LDL-cholesterol, QT interval, the prevalence of minor T wave abnormalities. All showed statistically significant, positive associations with spatial T wave axis quartile. Age was not associated with spatial T wave axis deviation. Table 1 Characteristics of the study population at baseline examination by spatial T wave axis extreme quartiles. The ARIC Study. Mean (SD) and percentages Variable (units) Spatial T wave axis deviation quartile Women , n = 7143 Men , n = 5133 T axis quartile 1: 0.33–14.75° T axis quartile 4: 30.20–60.76° T axis quartile 1: 0.19–16.19° T axis quartile 4: 30.41–60.88° Age (years) 53.5 (5.7) 53.7 (5.6) 54.4 (5.7) 54.4 (5.7) African-American % 18.4 41.7 13.0 32.1 Height (cm) 162.3 (5.9) 161.9 (6.0) 176.5 (6.2) 175.5 (6.7) Weight (Lb) 151.7 (32.5) 168.6 (37.8) 182.3 (30.8) 190.6 (31.8) BMI (kg/mm) 26.2 (5.4) 29.2 (6.3) 26.6 (4.1) 28.1 (4.3) WHR 0.88 (0.08) 0.90 (0.08) 0.96 (0.06) 0.96 (0.05) Smokers % 26 21 32 29 SBP (mmHg) 116.8 (18.2) 122.9 (19.4) 118.8 (15.5) 126.1 (18.5) DBP (mmHg) 70.3 (10.3) 74.2 (11.1) 73.3 (9.8) 78.2 (11.6) HTN % 25 43 22 44 Glucose (mmol/L) 5.8 (2.3) 6.1 (2.5) 5.8 (1.4) 6.2 (2.3) Diabetes % 8 13 7 14 HDL-C (mmol/L) 1.5 (0.4) 1.5 (0.4) 1.2 (0.4) 1.2 (0.4) LDL-C (mmol/L) 3.5 (1.0) 3.6 (1.0) 3.6 (1.0) 3.6 (1.0) Total Cholesterol (mmol/L) 5.6 (1.1) 5.7 (1.1) 5.4 (1.0) 5.5 (1.0) Tri-glyc. (mmol/L) 1.3 (0.8) 1.4 (1.0) 1.6 (1.2) 1.7 (1.1) QRS axis (degrees) 52.5 (30.0) 32.8 (32.0) 52.1 (34.3) 24.0 (35.9) Cornell voltage (uV) 926.1 (400.6) 1188.9 (437.4) 1165.6 (44.7) 1489.8 (490.6) QT interval (ms) 394.2 (27.4) 403.9 (28.3) 395.0 (29.2) 400.1 (29.8) Heart rate (bpm) 70.2 (9.8) 67.7 (9.9) 62.3 (10.3) 66.7 (10.1) QTc (ms) 399.2 (18.5) 403.9 (19.1) 415.0 (16.4) 418.5 (18.7) T axis frontal plane (degrees) 61.5 (12.3) 38.3 (25.4) 62.2 (12.5) 31.4 (26.8) Minor T wave abn.% 5.3 16.4 3.8 16.8 BMI = Body Mass Index, WHR = waist-to-hip-ratio, QTc = QT interval corrected for heart rate. bpm = beats per minute. SBP, DBP systolic and diastolic blood pressure, HTN, hypertension Of particular relevance is the association of the spatial T wave axis with hypertension and with the heart rate. The relationship between the spatial T wave axis and hypertension status (according to the JNC-VII classification) is presented in Table 2 by gender and adjusted for age, height and weight. The more pronounced the hypertension status the larger the mean values for the spatial T wave axis. Among the 3860 individuals defined as hypertensive, the mean (SD) values for the spatial T wave axis adjusted for age, height and weight were higher among those with uncontrolled HTN compared to those having the blood pressure levels below the treatment goal: 26.73 (0.27) degrees vs. 25.90 (0.27) (p = 0.03). Table 2 Mean spatial T wave axis deviation adjusted for age, height and weight, by JNC VII classification of blood pressure and gender. The ARIC study JNC VII BP stage Women Men N (%) Mean T axis (SE) N (%) Mean T axis (SE) Normal 3765 (52.73) 9.86 (0.10) 2373 (46.44) 11.23 (0.11) Pre-HTN 2286 (32.02) 18.29 (0.10) 1911 (37.40) 19.80 (0.11) Stage 1 HTN 841 (11.78) 25.81 (0.10) 648 (12.68) 26.63 (0.11) Stage 2 HTN 248 (3.47) 39.04 (0.10) 178 (3.48) 38.13 (0.1) We found a small but significant inverse association between spatial T wave axis deviation and heart rate. From the first to the fourth T wave axis quartile the mean (SE) values for the heart rate were: 70.24 (0.30), 69.05 (0.23), 69.20 (0.23), 67.70 (0.23) beats per minute for women and 67.26 (0.28), 65.74 (0.28), 65.56 (0.28), 66.67 (0.28) beats per minute for men. Differences between CHD cases and non-cases Compared to those who remained free of disease, men and women who subsequently developed CHD tended to have higher mean age, blood pressure, glucose and atherogenic lipids at baseline. They also were more likely to be smokers, have hypertension, diabetes, a higher Cornell voltage, minor T wave abnormalities and a lower mean values for the QRS axis in frontal plane. The mean QT interval and the mean T wave axis in frontal plane were not significantly different between cases and non-cases. In contrast to men, women who developed CHD had higher mean values for body mass index, waist to hip ratio, and heart rate at baseline. Spatial T wave axis deviation was only slightly increased among cases compared to non-cases (25.2 degrees versus 23.2 degrees among women, and 24.4 degrees versus 23.9 degrees among men). Mean values for the QT interval, heart rate, or T wave axis in frontal plane were not statistically different. Incident CHD events and relative risks of CHD Overall, the cumulative incidence of CHD was almost uniform across the distribution of the spatial T wave axis deviation, with an increase in the last quartile. The incidence rate was 4.26, 4.18, 4.28 and 5.62 per 1000 person-years across quartiles of spatial T wave axis deviation respectively. As Table 3 illustrates, the incidence rate ratio was 1.62 (95%CI of 1.15 to 2.27) for women in the upper quartile of the spatial T wave axis deviation compared with women in the bottom quartile. Adjustment for age, height and weight attenuated this ratio to 1.44 (95%CI of 1.02 to 2.04). Among men, the incidence rate ratio comparing the highest with the lower quartile was 1.09 (0.83–1.42), which was unchanged (1.08, 0.83–1.42) after adjustment for age, height and weight. Table 3 Cumulative incidence, incidence density rates and rate ratios for coronary heart disease at 12-years follow-up by spatial T wave axis quartile by gender. The ARIC study Risk estimate Spatial T wave axis deviation quartile among women T axis quartile 1: 0.33–14.75° T axis quartile 2: 14.75–22.38° T axis quartile 3: 22.39–30.32° T axis quartile 4: 30.33–60.88° Number of event-free participants at baseline 1787 1788 1784 1784 Number of CHD Events 54 57 53 86 Cumulative Incidence [%] 3.02 3.19 2.97 4.82 Person-years 21168 21176 21108 20838 Unadjusted Incidence Rate per 1000 person-years (95% CI) 2.56 (1.87–3.23) 2.70 (1.99–3.39) 2.51 (1.83–3.19) 4.13 (3.25–4.99) Unadjusted Incidence Rate Ratio (95% CI) 1 (referent) 1.05 (0.72–1.53) 0.98 (0.67–1.44) 1.62 (1.15–2.27) Adjusted Incidence Rate Ratio 1 (referent) 1.03 (0.71–1.50) 0.93 (0.64–1.36) 1.44 (1.02–2.04) Risk estimate Spatial T wave axis deviation quartile among men T axis quartile 1 0.19–16.19° T axis quartile 2 16.19–21.77° T axis quartile 3 21.77–30.40° T axis quartile 4 30.19–60.76° Number of event-free participants at baseline 1281 1276 1279 1277 Number of CHD Events 104 92 96 111 Cumulative Incidence [%] 8.12 7.21 7.51 8.69 Person-years 14699 14605 14603 14410 Unadjusted Incidence Rate per 1000 person-years (95% CI) 7.08 (5.72–8.44) 6.30 (5.01–7.59) 6.57 (5.26–7.89) 7.70 (6.27–9.13) Unadjusted Incidence Rate Ratio (95% CI) 1 (referent) 0.89 (0.67–1.18) 0.93 (0.70–1.23) 1.09 (0.83–1.42) Adjusted* Incidence Rate Ratio 1 (referent) 0.90 (0.68–1.20) 0.94 (0.72–1.25) 1.08 (0.83–1.42) * adjusted for age, height and weight Cox regression-modeling of the unadjusted association between CHD and spatial T wave axis deviation among women showed that for every 10 degrees increase in the spatial T wave axis deviation, there was a 1.16 (1.04 and 1.28)-fold increase in the risk of CHD (Table 4 ). When treating spatial T wave axis deviation categorically, borderline and abnormal deviation was associated with a 1.59 (1.20–2.10) and 1.69 (1.04–2.75)-fold higher risk of developing CHD compared to the normal category. When adjusting for age, height, weight, smoking, hypertension, diabetes mellitus, QRS axis and T wave minor abnormalities, this association for women was no longer statistically significant (hazard rate ratio of 1.03 (0.92–1.14). Neither heart rate or the QT interval (regardless of the formula used for heart rate correction), did qualify as confounders, as they were not associated with incident events in this study population. Table 4 Hazard rate ratios (95%CI) of coronary heart disease at 12 years of follow-up for ten degrees increase in the spatial T wave axis deviation and for the comparison of the borderline and/or the abnormal categories with the normal spatial T wave axis category. Data stratified by gender. The ARIC study Model Women N = 7143 Men N = 5113 Continuous T wave axis deviation* Categorical T wave axis deviation** Continuos T wave axis deviation* Categorical T wave axis deviation** Crude 1.16 (1.05–1.28) 1.59 (1.20–2.10) 1.69 (1.04–2.75) 1.05 (0.96–1.15) 1.11 (0.89–1.40) 1.27 (0.79–2.04) Adjusted for age (55 yr) 1.15 (1.05–1.27) 1.58 (1.19–2.09) 1.64 (1.01–2.67) 1.05 (0.96–1.15) 1.10 (0.88–1.39) 1.23 (0.76–1.98) Adjusted for age, height and weight 1.11 (1.00–1.23) 1.46 (1.10–1.94) 1.49 (0.92–2.44) 1.05 (0.96–1.15) 1.10 (0.87–1.39) 1.22 (0.75–1.97) Adjusted for age, height, weight and QRS axis 1.10 (1.00–1.22) 1.44 (1.09–1.92) 1.47 (0.90–2.40) 1.03 (0.94–1.13) 1.07(0.84–1.35) 1.17(0.72–1.89) Adjusted for age, height, weight, QRS axis and smoking 1.10 (1.00–1.22) 1.49 (1.12–1.99) 1.42 (0.87–2.32) 1.02 (0.93–1.12) 1.04(0.82–1.32) 1.09 (0.67–1.76) Adjusted for age, height, weight, QRS axis, smoking, HTN, and DM 1.04 (0.94–1.15) 1.32 (0.99–1.76) 1.06 (0.64–1.77) 0.95 (0.86–1.04) 0.93(0.73–1.18) 0.85(0.52–1.39) * Spatial T wave axis deviation was treated as a continuous variable and risk estimates are expressed for ten degrees (approximately 1 standard deviation in the distribution) increase in the spatial T wave axis deviation. Spatial T wave axis deviation was treated as a categorical variable and risk estimates are expressed for the comparison of the borderline category (30–45 degrees) to the normal category in the first row of the cell and for the comparison of the abnormal category (≥45 degrees) to the normal category (≤30 degrees) in the second row of the cell. * HTN and DM indicate hypertension and diabetes mellitus respectively. Hypertension status was defined as systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg or current use of antihypertensive medication. Diabetes status was defined as fasting glucose ≥126 mg/dL, nonfasting glucose ≥200 mg/dL, or a physician diagnosis or pharmacological treatment for diabetes. Among men, there was no significant association between spatial T wave axis deviation and CHD events. This lack of association persisted after adjusting for covariates. T wave axis in the frontal plane was not associated with coronary events either; the crude associations were nil and persisted as such in multivariate analysis in both women and men. When using the same T wave axis deviation categories as in the CHS and Rotterdam studies, borderline and abnormal deviation was associated with a 1.11 (0.89–1.40) and 1.27 (0.79–2.04)-fold higher risk for developing CHD compared to the normal category. After adjusting for age, height, and weight and as well for smoking, hypertension, diabetes, QRS axis and T wave minor abnormalities, the association remained statistically non-significant. The prevalence of an abnormal spatial T wave axis deviation (larger than 45 degrees) in the ARIC population was extremely low (4.5%) so these risk estimates need to be interpreted with caution. Minor T wave axis abnormalities were only slightly associated with the risk of developing a coronary heart disease event, in both men and women. The hazard rate ratios for CHD in relation to minor T wave abnormalities were 1.96 (1.37–2.81) for women and 1.31 (0.92–1.88) for men. After adjustment for age, height, weight, smoking, hypertension, diabetes and QRS axis, these estimates became 1.25 (0.86–1.80) for women and 1.02 (0.71–1.47) for men. The measured (uncorrected for heart rate) QT interval was not associated with coronary events, in either gender group. Incidence rates for coronary events in the first and the last gender-specific QT interval quartiles were 8.04 and 8.00 per 1000 person-years among men and 3.8 and 4.5 per 1000 person-years among women. Adjustment for age and heart rate (either by using the heart rate corrected QT interval by Bazett's formula, QT Index or by including heart rate in the Cox proportional models) did not change this lack of association. Discussion This prospective, population-based, bi-ethnic study of men and women free of coronary heart disease at baseline shows that spatial T wave axis deviation is not associated with incident coronary events during long-term follow-up. The QT interval was not associated, and minor T wave abnormalities were only weakly associated, with the incidence of coronary events after adjustment for various covariates. Given the lack of association between spatial T wave axis and coronary events in this study, several issues must be considered. Potentially, incomplete ascertainment or misclassification of CHD events could bias the results and obscure an underlying association. The excellent quality control strategies employed by the ARIC study, the highly standardized protocol for data collection, independent case ascertainment through the cohort follow-up, and the epidemiologic surveillance system in place at each of the four ARIC study communities make the possibility of misclassification of coronary heart disease very unlikely. Inadequate statistical power to detect a meaningful association is not likely, due to the large sample size, long follow-up and relatively large number of events. The minimum effect size that the present study could have missed was computed by dichotomizing spatial T wave axis deviation, then contrasting "borderline" and "abnormal " deviation with the "normal" category (nQuery Advisor, version 4.0 [ 36 ]). Given the observed distribution of T wave axis deviation in the present study, a log-rank test of the survival curves at 12 years of follow up would have approximately 79 to 99% power to detect differences between the survival curves corresponding to hazard ratios between 1.11 and 1.22, at a 0.05 two-sided significance level. Our results do not confirm the findings of the Rotterdam study [ 23 ] and the CHS [ 24 ] with regard to the spatial T wave axis. The risk of CHD associated with abnormal deviation (more than 45 degrees) versus normal T wave axis deviation (less than 30 degrees) was 2.9 (95% CI 2.0–4.3)) in the Rotterdam study and 1.58 (95%CI 1.25–1.99) in the CHS study. In contrast, findings from the present study are similar to those from the Multiple Risk Factor Intervention Trial (MRFIT) [ 25 ], which did not find a significant association between spatial T wave axis deviation at baseline and incident CHD events, although there was a significant relation between change in spatial T axis and long-term mortality from CHD. The discrepancy between our results and those reported in previous articles has several potential explanations, involving true variation between study populations (differences between study populations in terms of exposure distribution, demographic characteristics, absolute cardiovascular risk level or effect modification by genetic or environmental factors) and different patterns of confounding by other cardiovascular risk factors. Compared to participants in the present study, those in studies that found a significant association between T wave axis deviation and CHD were older (Rotterdam study and CHS), and more likely to have a history of myocardial infarction, angina pectoris (Rotterdam study) or silent (ECG-diagnosed) myocardial infarction and hypertension at baseline (CHS study). These study populations also had more ECG evidence of ischemia and infarction as reflected by the presence of Q-waves and major ST-T abnormalities (Rotterdam study) or T wave inversion (CHS study) but this was not accounted for these confounders in the analysis. By contrast, the MRFIT study and the present ARIC study excluded these participants, preserving only those with minor T wave abnormalities, and found only weak or non-significant associations with CHD events. Studies reporting stronger associations also had shorter follow-up time. To explore the potential impact of these dissimilarities, we performed several post hoc analyses under different scenarios. i) without excluding any participants In comparing individuals without CHD at baseline with prevalent CHD cases at baseline, the spatial T wave axis deviation was significantly different: 26.66 (16.81) and 44.88 (27.29) degrees respectively (p value < .0001). The entire distribution of the spatial T wave axis deviation among those with CHD at baseline was shifted to the right of the distribution of participants without CHD at baseline. This difference persisted statistically significant even after adjustment for age and several standard cardiovascular risk factors (anthropometry, smoking, hypertension, diabetes, heart rate, QRS axis and QT interval) with adjusted mean values of 26.79 and 41.47 degrees respectively. However, among those without CHD at baseline in our study, there were various conditions or markers of clinical interest, some of established predictive value, such as ST segment depression, negative T waves or atrial fibrillation. In the clinical setting, the need for improved prediction and discriminatory ability of a new potentially useful marker is particularly useful in the absence of such markers. After excluding Minnesota codes representing secondary repolarization abnormalities, this difference was markedly attenuated, with mean spatial T wave axis deviation of 23.84 (12.36) for those free of CHD and 24.08 (15.34) degrees for those with CHD. This suggests that spatial T wave axis has no ability to discriminate between CHD cases and CHD-free individuals beyond and above secondary repolarization abnormalities. This might be one of possible explanations for the discrepant results between different cohort studies exploring T wave axis, as different studies have used different exclusion criteria. As an alternative to the exclusion of Minnesota codes reflecting secondary repolarization abnormalities, we performed a separate analysis this time including QRS duration as a covariate. The relationship of the spatial T wave axis deviation with incident CHD events followed the same pattern as in the restricted dataset, i.e. a slight increase in the incidence rates in the fourth quartile of the T wave axis, for each gender. The crude hazard rate ratios (95%CI) for incident CHD events for a 10 degree increase in the spatial T wave axis were 1.18 (1.13–1.23) for women and 1.14 (1.09–1.19) for men. After adjustment for the same covariates as in the restricted data set and for the QRS duration, the hazard rate ratios (95%CI) were 1.05 (0.99–1.16) and 1.04 (0.98–1.1). ii) restricted to fatal events only Ventricular repolarization abnormalities are closely related to the propensity for arrhythmia. Although none of the published population-based studies on T wave axis deviation attempted to ascertain arrhythmic events, it is plausible that the arrhythmic component of the CHD events was the driving force of the observed associations in some published studies. This suggestion is supported by the fact that in both, Rotterdam and CHS study, the association between T wave axis and coronary events was stronger for fatal than non-fatal events. Similarly, the spatial QRS-T angle was recently reported to be associated with fatal but not with non-fatal cardiac events [ 37 ]. To explore this issue we restricted our analysis to fatal coronary events. During an average follow-up time of 11.6 years, 143 fatal events were recorded in our study population, 62 among women and 81 among men, with an overall cumulative incidence of 1.17%. The relationship of fatal events with the spatial T wave axis deviation followed the same pattern as the combined outcome, i.e. a slight increase in the incidence rates for CHD only in the fourth quartile of the spatial T wave axis deviation, for each gender. As expected, in the multivariate models (Table 5 ), the crude hazard rate ratios for fatal events were slightly larger than for the combined outcome. After adjustment for a limited number of risk factors (due to the small number of events), these modest associations were no longer statistically significant. However, these patterns must be interpreted with caution. While in the Rotterdam and CHS studies, the incidence rate for fatal events was 4.2 (95% CI 3.3–5.1) and 5.8 (95% CI 5.0–6.7) per 1000 person year respectively, this rate was much lower in our study: 1.01 per 1000 person-years (95% CI, 0.84–1.17). Table 5 Hazard rate ratios (95%CI) for fatal and combined, fatal and nonfatal coronary heart disease events at 12 years of follow-up for ten degrees increase in the spatial T wave axis deviation. Data stratified by gender. The ARIC study. Model Fatal + non-fatal events Fatal events Women Men Women Men Crude 1.16 (1.04–1.29 1.05 (0.96–1.15) 1.27(1.04–1.55) 1.24 (1.02–1.50) Adjusted* 1.06 (0.95–1.17) 0.96 (0.88–1.06) 1.11 (0.91–1.35) 1.09 (0.90–1.33) *Adjusted for weight smoking HTN and DM iii) similarly, we repeated the analysis without excluding those with negative T waves corresponding to MC 5.2, restricted to the age group greater than 55 years, restricted to participants with both hypertension and diabetes at baseline, restricted to CHD events occurring after a shorter follow-up (4–6 years). While the incidence rates were larger in absolute value in each T wave axis quartile, in relative terms no association with CHD events was detected. Experimental models to link the spatial T wave axis deviation with coronary evens are still sparse, but it can be speculated that an abnormal spatial T wave axis may possibly reflect disturbances in the repolarization process caused by subclinical myocardial disease, with or without an increased propensity for arrhythmic events, and thus an increased risk for fatal cardiac events. This is supported by the fact that subclinical disease is likely more prevalent in the older population of the CHS and Rotterdam study (which found an association between T wave axis and incident coronary events) than in the younger populations of the ARIC and MRFIT studies (both reporting a lack of association). Some indicators of subclinical disease such as the left ventricular hypertrophy (LVH) are clearly more prevalent in the Rotterdam than in the ARIC study. The presence of detected or undetected subclinical disease (LVH, patchy myocardial fibrosis) may lead to ventricular electrical instability and a higher proportion of cardiac death related to primary arrhythmic events in a population. Thus, measured or unmeasured, subclinical disease may play a confounding role, and suggests one more explanation for the discrepancy of results between different studies. The ARIC, CHS and MRFIT study protocols all used the same definition of the spatial T wave axis deviation, while the Rotterdam study used the frontal plane T wave axis. There are no data available to explain the discrepancy of results solely on the basis of different measurement of the T wave axis. The comparison of spatial T wave axis deviation with its temporal counterpart, the QT interval raises an intriguing question. Incident coronary events were strongly associated with T wave axis in the cohort of older adults of the Rotterdam study, which examined a population of older adults in whom a prolonged QT interval was previously reported to be predictive. In the current work, neither spatial T wave axis deviation nor the QT interval (as measured or heart-rate corrected by different formulae) was associated with incident coronary events. In our study, a statistical interaction between QT interval and spatial T wave axis deviation was not detected within the range of the QT interval in this population (data not shown). It is important to mention that in the Rotterdam study the mean QT interval (measured with identical methodology as in our study) was considerably larger than in the present study. From electrophysiological point of view, an abnormal ventricular repolarization can alter the QT interval, the T wave axis or both. It is therefore possible that a given magnitude of an altered spatial T wave axis in the presence of a QT interval within "the normal" ranges, may not be sufficient to elicit any causal association with incident coronary events. Our study found an inverse association between spatial T wave axis deviation and heart rate. Heart rate influences have been reported on the frontal plane T wave angle [ 18 ] and on the spatial ST-T vector [ 38 ]. The physiologic explanation of the dependency of an angular measure on heart rate is not clear. It is plausible that this phenomenon is similar to some degree with that observed for another index of altered repolarization, namely T wave alternans, characterized by the change in amplitude, morphology and axis of the spatial T wave and associated with an increased risk of sudden cardiac death. Even if the mechanism of heart rate dependency of the spatial T wave axis remains elusive, it raises questions about the need for "rate correction" of this measurement. Our study confirms the results of other studies [ 18 , 19 ] in finding a strong association between spatial T wave axis and hypertension status. The explanation is likely related to the presence of an increased left ventricular wall in the context of the left ventricular hypertrophy. It is also possible that increased spatial Twave axis deviation is related to other processes present in the hypertensive myocardium, such as microfocal areas of fibrous tissue and/or increased alteration of ionic channels. These findings suggest that spatial T wave axis deviation may serve as an auxiliary early marker of repolarization abnormalities in hypertensive individuals. Due to the low prevalence of left ventricular hypertrophy in our study population, we were not able to further explore this issue. Strengths and limitations The present study has several strengths. The highly standardized data collection procedure and ECG protocol increase the internal validity of the findings. The ECG recording was performed by trained technicians using a standardized protocol for lead placement. The events ascertainment in the ARIC study is ensured by a highly standardized quality assurance protocol. The inclusion of apparently healthy participants in a population-based study, of African American and white men and women, the large sample size and the geographic distribution of the study population enhance generalizability. This study also has some limitations. The narrow age range precluded an extensive investigation of the role of age. The study also was limited by the lack of accurate measurements for characterizing the elliptical shape of the thoracic cavity. Constitutional and anthropometric variables such as the chest transverse diameter or chest shape have been associated with a shift of the T wave axis [ 39 ] or changes in average T wave potential amplitudes. A slender body with a limited spatial sampling of the thorax directly overlaying the heart can cause lower amplitudes in women than in men [ 40 ]. While our study attempted to account for this fact by adjusting for height and weight, a more accurate measurement of thoracic cavity, shape or body surface area would probably result in a better ascertainment of spatial T axis. Nonetheless, previous studies reporting positive findings did not account for the shape of thoracic cavity either, and it is unlikely that such measurement would have influenced the findings of the present study. Ventricular replarization is a complex process, which cannot be fully captured by only a limited number of indexes such as QT interval and spatial T wave axis, exploring either the temporal aspects or the general direction of this process. Other indexes or more complex descriptors of the T wave loop have been described to be accurate markers of an altered repolarization [ 41 ]. The spatial QRS-T angle has recently regained attention and several studies have attested its usefulness in post infarction [ 12 ] and hypertensive patients [ 19 ]. The spatial QRS-T angle was also the strongest predictor of fatal cardiac events, even after controlling for the frontal plane T axis, in the large population-based cohort of men and women aged 55 years or older of the Rotterdam study [ 37 ]. Unfortunately, this index is not currently available in our database. Conclusions In conclusion, this prospective, population-based, bi-ethnic study of men and women free of coronary heart disease at baseline shows that spatial T wave axis deviation is not associated with incident coronary events during long-term follow up. It is doubtful that T wave axis deviation would add benefit in the prediction of CHD events above and beyond the current traditional risk factors. The QT interval was not associated with incident coronary events, and minor T wave abnormalities were only weakly associated among women. Competing interests The author(s) declare that they have no competing interests. Authors' contributions GV performed the overall study design, statistical analysis and manuscript preparation. PR designed the data collection protocol, participated in the interpretation of the ECG data, provided comments on the manuscript. RP participated in the quality control of the ECG data, participated in the interpretation of the findings and with comments on the manuscript. EAW participated in the interpretation of the findings and with comments on the manuscript, provided assistance in the manuscript preparation. LEC provided oversight of the statistical analysis, verified the appropriateness of statistical models, provided assistance in the manuscript preparation. ARF participated in the interpretation of the findings and with comments on the manuscript, provided assistance in the manuscript preparation. WDR collaborated in the study design, participated with comments on the manuscript. ZMZ managed the automated coding of ECGs and the quality assurance. RC participated with comments on the manuscript. GH collaborated in the design of the study, provided comments on the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546201.xml
526287	Fully human IgG and IgM antibodies directed against the carcinoembryonic antigen (CEA) Gold 4 epitope and designed for radioimmunotherapy (RIT) of colorectal cancers	Background Human monoclonal antibodies (MAbs) are needed for colon cancer radioimmunotherapy (RIT) to allow for repeated injections. Carcinoembryonic antigen (CEA) being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs. Methods XenoMouse ® -G2 animals were immunized with CEA. Among all the antibodies produced, two of them, VG-IgG2κ and VG-IgM, were selected for characterization in vitro in comparison with the human-mouse chimeric anti-CEA MAb X4 using flow cytometry, surface plasmon resonance, and binding to radiolabeled soluble CEA and in vivo in human colon carcinoma LS174T bearing nude mice. Results Flow cytometry analysis demonstrated binding of MAbs on CEA-expressing cells without any binding on NCA-expressing human granulocytes. In a competitive binding assay using five reference MAbs, directed against the five Gold CEA epitopes, VG-IgG2κ and VG-IgM were shown to be directed against the Gold 4 epitope. The affinities of purified VG-IgG2κ and VG-IgM were determined to be 0.19 ± 0.06 × 10 8 M -1 and 1.30 ± 0.06 × 10 8 M -1 , respectively, as compared with 0.61 ± 0.05 × 10 8 M -1 for the reference MAb X4. In a soluble phase assay, the binding capacities of VG-IgG2κ and VG-IgM to soluble CEA were clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10 -7 M was needed to precipitate approximatively 1 ng 125 I-rhCEA as compared with 10 -9 M for MAb X4, suggesting a preferential binding of the human MAbs to solid phase CEA. In vivo , 24 h post-injection, 125 I-VG-IgG2κ demonstrated a high tumor uptake (25.4 ± 7.3%ID/g), close to that of 131 I-X4 (21.7 ± 7.2%ID/g). At 72 h post-injection, 125 I-VG-IgG2κ was still concentrated in the tumor (28.4 ± 11.0%ID/g) whereas the tumor concentration of 131 I-X4 was significantly reduced (12.5 ± 4.8%ID/g). At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. A pertinent analysis of VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver. Conclusion Our human anti-CEA IgG2κ is a promising candidate for radioimmunotherapy in intact form, as F(ab') 2 fragments, or as a bispecific antibody.	Background During the last few years, radioimmunotherapy (RIT) using MAbs to specifically target therapeutic radiation doses to tumors has led to objective responses in radiosensitive hematological cancers, particularly, in non-Hodgkin's lymphoma (NHL) [ 1 , 2 ]. On the basis of these clinical results, ibritumomab tiuxetan ( 90 Y-Zevalin; IDEC Pharmaceuticals) was registered for treatment of relapsed, indolent, and transformed CD20 + NHL and, more recently, tositumomab ( 131 I-Bexxar; Corixa) received regulatory approval; development of other promising products is in the pipeline [ 3 ]. Although targeting of solid tumors with radiolabeled antibodies was first reported years ago [ 4 , 5 ], RIT success in such tumors has been limited to patients with stable disease, occasional mixed responses, and serological responses [ 6 - 8 ]. Different parameters can be considered as responsible for these results: (i) the decreased radiosensitivity of solid tumors as compared with hematological cancers [ 9 , 10 ], (ii) the difficult penetration of MAbs in solid tumors [ 11 ], and (iii) consequently, the limited radiation dose that can be delivered to the tumor [ 12 , 13 ]. However, recent studies have reported a therapeutic window for RIT in solid tumors in small-volume and minimal residual disease [ 8 ] and in combination with chemotherapy [ 14 ]. The authors of all the recent pertinent clinical studies agree with the need of repeated injections for RIT of solid tumors and, consequently, with the need of humanized or, preferentially, human MAbs [ 14 , 15 ]. Colorectal cancers represent a high percentage of solid tumors and are dramatically in need of therapeutic progress. Surgery is the only potentially curative treatment. Despite recent developments in chemotherapy protocols, the overall median survival in metastatic colorectal cancer remains inferior to two years, and the recurrence rate after resection of a stage III tumor is up to 50% [ 16 - 18 ]. For RIT of colorectal cancers, carcinoembryonic antigen (CEA) is a preferential target antigen since (i) it is expressed in almost all tumors (>95%), (ii) it is available at high antigenic density on the cell surface, and (iii) many clinical studies have demonstrated a low MAb uptake in normal intestine despite CEA expression on these tissues. The only limitation of CEA as target antigen in RIT is the possible presence of circulating CEA in the serum of cancer patients, but this is without consequence in small-volume and minimal residual disease in which its level is generally low [ 19 ]. Different chimeric or humanized anti-CEA MAbs have been described and evaluated in experimental and clinical studies [ 8 , 14 , 15 , 19 , 20 ]. In the present study using the XenoMouse ® technology, we describe the generation and the characterization of two fully human anti-CEA antibodies, one IgG2κ and one IgM, designed for RIT of colorectal cancers. Methods Generation of fully human MAbs from XenoMouse ® strains Generation and characterization of the XenoMouse ® -G2 strain, engineered to produce fully Human IgG2κ antibodies, was described by Mendez et al. [ 21 ]. XenoMouse ® -G2 animals were immunized i.p. with 20 μg of human recombinant CEA (rhCEA) [ 22 ] emulsified in complete Freund's adjuvant for the primary immunization and in incomplete Freund's adjuvant for additional immunizations carried out at one month intervals. Immunization was repeated three to five times. Two days before fusion, mice were boosted i.v. with 100 μg rhCEA in phosphate buffered saline (PBS). Spleen cells from immunized mice were fused with the non-secretory myeloma P3-X63-Ag.8.653 by addition of polyethylene glycol (PEG) and were subjected to HAT selection. Wells containing growing cells were evaluated for the production of the desired antibody, and if positive, the cultures were cloned. The hybridomas described in this report were subcloned at least five times. Reference anti-CEA MAbs and control MAb The mouse-human chimeric MAb X4 was used as positive control in all the experiments. MAb X4 was constructed using the variable domains from the murine MAb CE25 and the constant domains from a human IgG 4κ subclass [ 23 , 24 ]. It is specific for the CEA epitope Gold 4 [ 24 ] and does not cross-react with NCA or other granulocyte proteins [ 25 ]. Chimeric MAb X4 was produced in Sp2/0 cells transfected with a single vector containing both the chimeric heavy and light chains [ 24 ]. Murine MAbs F6, 35A7, B17, CE25, and 192, which are specific for the CEA epitopes Gold 1 to 5, respectively, were used for epitope determination [ 26 ]. MAb F6 was kindly provided in purified form by Schering-CIS Biointernational (Gif-sur-Yvette, France). MAbs 35A7, B17, CE25, and 192 were produced from mouse hybridoma ascites fluid by ammonium sulfate precipitation and ion-exchange chromatography. The human IgG MonoD, kindly provided by MAbgène (Alès, France), was used as irrelevant human IgG 1 [ 27 ]. Cell lines and human granulocytes The CEA-positive human colon carcinoma LS174T cell line [ 28 ] was obtained from the Cell Distribution Center, American Type Culture Collection (Rockville, MD). The CO115-5F12 clone, obtained by transfection of the full-length CEA-cDNA in a CEA negative clone of the CO115 human colon carcinoma cell line, has been described [ 29 ]. Cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, streptomycin (0.1 mg/ml), penicillin (0.1 IU/ml), and amphotericin B (0.25 μg/ml). The neomycin analogue G418 was added at a concentration of 200 μg/ml to the CO115-5F12 cell culture. All culture medium supplements were purchased from Life Technologies, Inc. (Gibco BRL, Gaithersburg, MD). For flow cytometry analysis, cells were harvested after incubation for a few minutes in trypsin-EDTA (0.5 mg/ml and 0.2 mg/ml, respectively). Granulocytes were obtained from heparin-treated human peripheral blood by using gradient density centrifugation methods. A double gradient was formed by layering an equal volume of HISTOPAQUE ® -1077 and HISTOPAQUE ® -1119. Following centrifugation at 700 g for 30 minutes, cells of the granulocytic series were found at the 1077/1119 interphase. Screening by ELISA and flow cytometry The specificity of the antibodies in hybridoma supernatants was determined by ELISA using rhCEA to capture the antibodies (coating overnight at 2 μg/ml rhCEA at room temperature). Horse radish peroxidase (HRP)-conjugated goat anti-human IgG κ (Sigma, Lyon, France) and HRP-conjugated sheep anti-human IgG (γ chain) (Silenius, Hawthorn, Australia) were used as detection antibodies. Determination of CEA or NCA specific antibodies in hybridoma supernatants was carried out by flow cytometry using CEA positive cells (CO115-5F12) and NCA positive cells (human granulocytes). About 5 × 10 5 cells were incubated for 1.5 h at 4°C with hybridoma supernatants or controls (RPMI medium for background measurement and RPMI containing 20 μg/ml MAb X4 for positive control). After washing, the cells were incubated with an FITC conjugated goat anti-human IgG kappa light chain (Sigma) or with the murine anti-human μ chain DA4-4 (ATCC HB-57), FITC labelled in our laboratory, for 1 h at 4°C; then they were washed twice before analysis on a FACScanII (Becton-Dickinson, Le-Pont-De-Claix, France). Each figure represents data obtained from analysis of 10000 cells. Human MAb production, purification, and molecular characterization The percentage of fetal calf serum was gradually reduced in the culture medium before MAb purification (10, 5, 2.5 and 0%). MAbs were purified from large volumes of hybridoma supernatants or ascites produced in nude mice using Hitrap ® NHS-anti-human κ chain MAb HP6053 (ATCC CRLC-1758). Proteins separated on SDS-PAGE 6% polyacrylmamide gels were transferred to a nitrocellulose membrane (Protran BA85, Schleicher and Schuell, Dassel, Germany). Non-specific binding sites were blocked overnight at 4°C by incubation with 5% (w/v) non-fat dry milk in TBS. The membrane was probed for 2 h at room temperature with serum diluted 1:1000 or the following antibodies: anti-human κ light chain-HRP conjugate (A7164, Sigma), anti-human λ light chain-HRP conjugate (A5175, Sigma), anti-human μ chain-HRP conjugate (A0420, Sigma), rabbit anti-human J chain-specific antiserum [ 30 ]. Bound serum antibodies were detected with a goat anti-rabbit whole molecule-alkaline phosphatase (AP) conjugate (A8025, Sigma). HRP was detected by addition of a chloronaphthol (Sigma) solution containing 0.05% of hydrogen peroxide and AP by addition of BCIP-NBT (Sigma). A human pentameric IgM anti-Rhesus D including a J chain, kindly provided by MAbgène (Alès, France), was used as positive control[ 27 ]. Antibody VH and Vκ cDNAs were recovered from hybridomas by RT-PCR and sequenced using the ABI-PRISM Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer, Boston, MA). Determination of V, D, and J gene usage was performed using in silico methods. Measurement of antibody affinity to CEA The affinities of the antibodies for CEA were determined by using surface plasmon resonance (SPR) technology (Biacore AB, Uppsala, Sweden). rhCEA [ 22 ] was immobilized on a CM5 sensor chip by the method of thiol ligation according to the manufacturer's instructions (BIACORE Methods Manual Supplement 5a). Each MAb was injected at a concentration of 50 μg/ml in HBS buffer (Hepes-buffered saline, pH 7.4, 3 mM EDTA ; 0.05% BIACORE surfactant) at a flow rate of 20 μl/min. Dissociation was carried out in running buffer (HBS). Regeneration of the sensor chip was performed by using 15 μl of 100 mM HCl. The kinetic parameters were determined by using BIAevaluation 3.2 software. MAbs and CEA radioiodination Batches of 50 μg or 100 μg of MAb or rhCEA were labelled with 4.6 MBq or 11.5 MBq, respectively, of 125 I or 131 I, kindly provided by Schering-CIS Biointernational by the iodogen method (1,3,4,6-tetrachloro-3α, 6α-diphenylglycoluryl, Sigma). Free radioiodine was separated from the protein on a Sephadex G-25 column (Pharmacia) equilibrated in PBS, pH 7.4. Binding of MAbs to CEA in a soluble phase assay About 1 ng 125 I-rhCEA (final concentration 16.7 × 10 -12 M) was incubated with increasing concentrations of antibody (68.3 × 10 -12 to 33.3 × 10 -15 M) for 2 h in 0.15 M phosphate buffer, pH 7.4. CEA-antibody complexes were precipitated at 4°C with 53.5% (v/v) saturated ammonium sulfate. Background binding to an irrelevant human IgG, MonoD, was subtracted. The radioactivity precipitated with a rabbit polyclonal anti-CEA serum was taken as 100%. Epitope mapping by RIA A competitive binding assay of radiolabeled human MAb and unlabeled anti-CEA MAb was used to determine the CEA epitope recognized by the human MAbs. RhCEA (100 ng/well) in TBS was coated on microtiter wells overnight at room temperature. An excess (500 ng/well) of each of the different reference anti-CEA MAbs, specific for the Gold epitopes 1 to 5, was then added to the wells and incubated for 1.5 h at 37°C. Then, without washing, each 125 I-human MAb (15 ng) was added to the wells and incubated for 1 h 30 at 37°C. The percentage of binding was determined by measuring the radioactivity bound to the rhCEA after two washings. Biodistribution studies Two million LS174T cells were grafted s.c. into the right flank of female Swiss nude mice (nu/nu, Iffa Credo, l'Arbresle, France). When the tumors had reached a volume of about 150 mm 3 (100 to 300 mm 3 ), mice were grouped according to tumor volume. Lugol iodine solution (10% solution) was added to the drinking water one day before the injection of radiolabeled MAbs. Groups of four mice were injected with a 125 I-labeled human MAb (VG-IgG2κ or VG-IgM) together with 131 I-labeled chimeric MAb X4 as positive control. The total amount of each injected antibody was adjusted to 8 μg protein by adding unlabeled MAb. To determine the biodistribution of the MAbs, mice were sacrificed 24 or 72 h after injection. The blood, tumor, and all normal organs were weighed, and the differential radioactivity was measured in a dual channel scintillation counter. The results are expressed as the percentage of the injected dose of radioactivity present per gram of tissue (% ID/g). Results Human anti-CEA MAb characterization In order to develop human anti-CEA MAbs, XenoMouse ® -G2 animals were immunized with rhCEA. The XenoMouse ® -G2 strain produces both fully human IgG2κ and fully human IgMκ antibodies as part of the normal immune response, as described by Mendez et al. [ 21 ]. Fusion of splenic B cells from immunized mice with mouse myeloma cells yielded a panel of hybridomas that secreted human anti-CEA antibodies as determined by ELISA and flow cytometry analysis (data not shown). These MAbs were produced for in vivo tumor targeting purposes. For this reason, (i) hybridoma supernatants were screened by flow cytometry because, by this technique, the selection is made on cell membrane-bound CEA, which is in a conformation as close as possible to that observed in vivo ; and (ii) hybridoma supernatants were screened on human granulocytes to eliminate all hybridomas producing anti-NCA antibodies. Among the 52 antibodies produced, two, VG-IgG2κ and VG-IgM, were selected for further characterization based on hybridoma stability and flow cytometry analysis results. V H and V L domains sequencing confirmed the VG-IgG2κ and VG-IgM monoclonality and the isotype (data not shown). Flow cytometry analysis demonstrated strong binding of purified MAbs on CEA-expressing cells CO115-5F12 (Figure 1A ) without any binding on NCA-expressing human granulocytes (Figure 1B ). MAb VG-IgG2κ and VG-IgM epitope specificities were determined in a competitive binding assay using five reference MAbs directed against the Gold 1 to 5 CEA epitopes [ 26 ]. As demonstrated by this assay (Table 1 ), the two human MAbs were directed against the CEA Gold 4 epitope since they were only inhibited by the murine MAb CE25 and the chimeric MAb X4. A partial inhibition, attributed to steric hindrance, was observed with MAb B17 (Gold 3) for VG-IgG2κ (31%) and X4 (13%). VG-IgM was further analyzed for the presence of J chain using western blot. The J chain is a ligand for the poly-Ig receptor expressed on mouse hepatocytes. Figure 2 confirms that VG-IgM is constituted of human κ light chain, human μ heavy chain, and J chain as compared with an anti-Rhesus D IgM which also contains a J chain but is composed of a λ light chain [ 27 ]. MAb binding to solid phase CEA and to soluble phase CEA Using surface plasmon resonance technology, the affinities of purified VG-IgG2κ and VG-IgM were determined to be 0.19 ± 0.06 × 10 8 M -1 and 1.30 ± 0.06 × 10 8 M -1 , respectively, as compared with 0.61 ± 0.05 × 10 8 M -1 for the reference MAb X4. MAb binding to soluble phase CEA was measured using trace amounts of 125 I-CEA incubated with increasing concentrations of antibodies (Figure 3 ). The binding capacities of VG-IgG2κ and VG-IgM to soluble CEA were found to be clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10 -7 M was needed to precipitate around 1 ng 125 I-rhCEA (final concentration 16.7 × 10 -12 M) as compared with 10 -9 M for MAb X4 (Figure 3 ). Biodistribution studies The human MAbs were compared to the chimeric MAb X4 in nude mice bearing human colon carcinoma LS174T xenografts. Two groups of four mice were co-injected with 125 I-VG-IgG2κ and 131 I-X4. In the first group of mice dissected 24 h post-injection, 125 I-VG-IgG2κ demonstrated a high tumor uptake (25.4 ± 7.3% ID/g), very close to that of 131 I-X4 (21.7 ± 7.2% ID/g) (Figure 4 ). At 72 h post-injection, the 125 I-VG-IgG2κ was still concentrated in the tumor with 28.4 ± 11.0% ID/g whereas the tumor concentration of 131 I-X4 was significantly reduced, with only 12.5 ± 4.8% ID/g (Figure 4 ). This difference was attributed to a higher in vivo stability of VG-IgG2κ than X4 since the %ID recovered in the whole mouse at that time was 74.1 ± 1.5 and 55.6 ± 1.6 for the human and the chimeric MAbs, respectively. At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. At 24 h, the tumor-to-normal tissue ratios of the antibodies were in the same range for 131 I-X4, with representatives values of 10.98 ± 0.35, 28.78 ± 0.36, and 2.24 ± 0.38 for liver, muscle and blood, respectively, and for 125 I-VG-IgG2κ, which gave values of 8.63 ± 0.30, 28.30 ± 0.31, and 1.74 ± 0.32 for the same organs, respectively (Table 2 ). At 72 h, the increase in the tumor-to-normal tissue ratios was very similar for the two MAbs (Table 2 ). In two other groups of mice, 125 I-VG-IgM was co-injected with 131 I-X4. The reference chimeric MAb X4 gave tumor localization results comparable to that obtained when it was co-injected with 125 I-VG-IgG2κ (19.1 ± 1.9 and 11.9 ± 3.1% ID/g tumor at 24 h and 72 h, respectively). Due to the mouse poly-Ig receptor expression in liver, 125 I-VG-IgM was eliminated very rapidly, giving only 3.7 ± 1.0 and 0.3 ± 0.1% ID/g blood at 24 h and 72 h, respectively. This short clearance was responsible for a low tumor uptake (7.4 ± 2.8 and 1.8 ± 2.4% ID/g tumor at 24 h and 72 h, respectively). The percentage of injected MAb recovered in the whole mouse at 24 h was only 20.5% for 125 I-VG-IgM as compared with 53.7% for 131 I-X4. Discussion Monoclonal antibodies are now routinely used in the clinic. Whereas the first generation of MAbs were murine or chimeric antibodies, it is now clear that the best clinical results are obtained with humanized or fully human MAbs [ 31 ]. Fully human antibodies such as ABX-EGF are anticipated to exhibit a long serum half-life and minimal immunogenicity with repeated administration, even in immunocompetent patients [ 32 ]. In some situations, naked MAbs have demonstrated their therapeutic efficacy, particularly, when the target antigen is a receptor implicated in cell proliferation processes [ 33 , 34 ]. However, the addition of radioactive isotopes on already efficient MAbs can lead to improved therapeutic results like that obtained with the anti-CD20 antibodies [ 35 ]. RIT remains attractive for solid tumors where the antibody penetration is limited and where the cross-fire phenomenon could lead to the destruction of cells which were not targeted by the radiolabeled MAb [ 13 ]. According to all the published or ongoing clinical studies, RIT could be applied to micrometastases from solid tumors or solid tumors in the minimal residual disease states [ 6 , 8 , 14 , 15 ]. One potential limitation of intact human MAbs for RIT could be their long serum half-life, which could lead to bone marrow suppression. The use of human MAb fragments for RIT would reduce their serum half-life, and possibly circumvent this limitation. Among solid tumors, colorectal cancers represent one of the main causes of death, and CEA is well known as an ideal target antigen for RIT of these cancers [ 12 ]. Up to now, only a few human anti-CEA antibodies have been described. A human MAb directed against the carbohydrate moiety of CEA was described by Tsukazaki et al [ 36 ]. However, it was poorly characterized, and homology between the carbohydrate moieties of CEA and related molecules such as NCA would certainly lead to limited specificity of this anti-CEA MAb. Very recently, Imakiire et al. described human antibodies generated using the KM-Mouse [ 37 ]. They demonstrated complement- and cell-dependent cytotoxicity in vitro and presented preliminary data on tumor growth inhibition using MKN-45 cells grafted into SCID mice. However, they did not give any results on the biodistribution of their antibodies in radiolabeled form nor indications on how they could be used in RIT. Anti-CEA MAb PR1A3, which exhibits preferential binding to cell-bound CEA, was recently humanized, but to our knowledge, this MAb has not yet been evaluated in experimental or clinical studies [ 20 ]. A few clinical phase I or II trials suggest a certain degree of efficiency of humanized or chimeric anti-CEA mAbs, radiolabeled with either 131 Iodine or 90 Ytrium, in heavily pre-treated patients with metastatic colorectal cancer (MCRC) CT84.66 [ 8 , 14 , 15 ]. One of these trials showed a few objective tumor responses in MCRC of small-volume disease and provided some arguments in favor of this kind of therapy in an adjuvant setting [ 8 ]. The development of such chimeric, humanized, or human anti-CEA MAbs by different academic groups and industrial companies underlines the interest to generate a fully human MAb for RIT of colorectal cancers. In the present study, the characterization of our fully human anti-CEA MAbs was conducted in comparison with the chimerized anti-CEA MAb X4, which has been shown to be clinically relevant [ 19 , 24 ]. Furthermore, the newly developed human MAbs were found to be directed against the same CEA epitope, namely Gold 4 (Table 1 ). That makes X4 an even better positive control, in particular, for the affinity measurements; although no difference has been reported between the different CEA epitopes for tumor immunotargeting [ 26 ]. VG-IgG2κ and VG-IgM are CEA-specific, i.e., NCA negative. This is particularly important for VG-IgM since the avidity generated by the pentameric molecule could have resulted in enhanced non-specific binding to CEA-related molecules. The VG-IgG2κ and VG-IgM affinity constants were found to be similar to that of the control MAb X4 when determined using the BIACORE technology, but the binding of the human MAbs to soluble CEA (Figure 3 ) was clearly weaker than that of X4. This is particularly interesting for in vivo use in patients where some circulating CEA can be found. The molecular basis of this observation is not clear. The only possible comparison is with MAb PR1A3, which preferentially binds to cell bound CEA, and to a recombinant chimeric protein containing only the CEA B3 domain [ 20 , 38 ]. This reduced binding to the whole soluble CEA was attributed by the authors to be due to a conformational change supposed to occur when the CEA is shed into the circulation, resulting in steric blocking of antibody access to the B3 domain [ 20 ]. Using human colon carcinoma bearing nude mice, we obtained high tumor uptakes with VG-IgG2κ, making this antibody a good candidate for future clinical studies (Figure 4 ). In addition to the comparison with chimeric MAb X4, we also performed biodistribution studies comparing VG-IgG2κ and the murine MAb 35A7, with which we obtained the highest tumor uptakes in tumor bearing nude mice [ 39 , 40 ]. Seventy-two hours post injection, 125 I-VG-IgG2κ localized in the tumor up to 26.2 ± 1.7% ID/g as compared with 28.5 ± 2.8% ID/g for 131 I-35A7, suggesting a tumor residence time as long as that observed for MAb 35A7 (data not shown). These results could seem contradictory, given the fact that the affinity of VG-IgG2κ for CEA is not as high as that of Mab 35A7. This could be explained by the "affinity barrier" effect described in solid tumors by several authors who demonstrated that very high affinity MAbs localized at the periphery of tumor nodules and that lower affinity MAbs are able to distribute homogenously in these nodules [ 41 - 43 ]. Since, up to now, there are no data available on the biodistribution of any anti-CEA IgM in mice, we decided to analyze the biodistribution of our VG-IgM in LS174T tumor bearing nude mice. The disappointing tumor uptakes (7.4 ± 2.8 and 1.8 ± 2.4%ID/g tumor at 24 h and 72 h, respectively) could be attributed to a very short half-life due to poly-Ig receptor expression in the mouse liver which induces a rapid hepatobiliary transport of poly-IgA and IgM [ 44 , 45 ]. Based on the results obtained by Borchardt et al., we compared the tumor uptakes following i.p. and i.v. injection of the VG-IgM [ 46 ]. The tumor uptakes observed after i.p. injection were even lower (0.25%ID/g tumor at 24 h). These results are in contradiction with those obtained by Borchardt et al. [ 46 ]. In SK-Ov3 peritoneal carcinomatosis bearing nude mice, these authors showed a marked difference in tumor uptake between i.v. and i.p. injections of AC6C3-2B12 human IgM: 39% vs. 0.9%ID/g tumor for i.p. vs. i.v. injection, respectively, at 24 h and 28% vs. 1.4%ID/g at 48 h [ 46 ]. Liver and spleen uptakes were reduced following the i.p. injection as compared with the i.v. injection, but these uptakes in normal tissues could be related to this particular IgM and not relevant for our VG-IgM [ 46 ]. Indeed, the precise nature of the target antigen of their AC6C3-2B12 human IgM is unclear, but the discrepancy between our respective results could be due to the lack of J chain in their IgM. Without the J chain, IgM is unable to bind to the poly-Ig receptor, and as such it is not transported into the bile nor eliminated rapidly. The presence of the J chain in our VG-IgM makes it a fully functional IgM but limits its uptake by the human tumor grafted in nude mice. Conclusions In the present study, we described two fully human anti-CEA MAbs. Even though the results obtained in tumor bearing nude mice cannot be extrapolated directly to humans, VG-IgM remains attractive for RIT of CEA-positive peritoneal carcinomatosis in man [ 47 ]since humans lack hepatic expression of poly-Ig receptor [ 48 ]. A first step toward this aim will be to study the biodistribution and tumor uptakes of low doses of radiolabeled VG-IgM. VG-IgG2κ is obviously a candidate for radioimmunotherapy in intact form, as F(ab') 2 fragments, or as a bispecific antibody to be used in the affinity enhancement system (AES) approach [ 49 ]. Furthermore, we intend to test it in a model for immunophotodetection of cancer [ 50 ], and it could be the basis for preparing different anti-CEA immunoconjugates [ 51 , 52 ] and fusion proteins [ 53 ]. Competing interests No competing interest for VG, FG, EF, MY, MP and AP. RXL and BB are employees of Abgenix, Inc. Authors' contributions VG participated in the design of the study, performed all the cell fusions, cell culture experiments, antibody characterization assays and in vivo experiments. FG and EF participated in the cell culture experiments and antibody characterization assays. MP performed the affinity measurements using BIACORE. MY participated in the design of the study. RXL and BB sequenced the V H and V L domains. AP conceived the study, participated in its design and coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC526287.xml
517933	Tissue detection of natural killer cells in colorectal adenocarcinoma	Background Natural killer (NK) cells represent a first line of defence against a developing cancer; however, their exact role in colorectal cancer remains undetermined. The aim of the present study was to evaluate the expression of CD16 and CD57 [immunohistochemical markers of natural NK cells] in colorectal adenocarcinoma. Methods Presence of NK cells was investigated in 82 colorectal adenocarcinomas. Immunohistochemical analysis was performed, using 2 monoclonal antibodies (anti-Fc Gamma Receptor II, CD16 and an equivalent to Leu-7, specific for CD-57). The number of immunopositive cells (%) was evaluated by image analysis. The cases were characterized according to: patient gender and age, tumor location, size, grade, bowel wall invasion, lymph node metastases and Dukes' stage. Results NK cells were detected in 79/82 cases at the primary tumor site, 27/33 metastatic lymph nodes and 3/4 hepatic metastases; they were detected in levels similar to those reported in the literature, but their presence was not correlated to the clinical or pathological characteristics of the series, except for a negative association with the patients' age (p = 0.031). Conclusions Our data do not support an association of NK cell tissue presence with clinical or pathological variables of colorectal adenocarcinoma, except for a negative association with the patients' age; this might possibly be attributed to decreased adhesion molecule expression in older ages.	Background Colorectal adenocarcinoma is a neoplasm in which prognosis is mainly determined by the histological stage. However, the prognosis of certain patient groups, especially those of intermediate stages, remains vague [ 1 ]. The main weakness of the currently used prognostic markers in colorectal adenocarcinoma, is their inability to point out patients without metastases, whose clinical course will progress unfavorably and patients with metastatic disease who will have a relatively better outcome [ 1 ]. Therefore, development of new prognostic markers is essential, as they might help in the planning of more effective treatment modalities. Markers of the host's immune response could potentially be helpful in this field. Natural killer (NK) cells play a pivotal role in innate immunity and immunological surveillance. Their cytotoxic effects may be initiated without prior immunization and thus NK cells have been considered as a first line of defence of the host against a developing cancer [ 2 , 3 ]. In spite of their small presence in tissue samples from colorectal adenocarcinomas, NK cells manifest a potent cytotoxic anti-tumor effect [ 3 - 5 ]. Expression of various protein surface markers is essential for these cells to perform their activities [ 2 ]. Among these markers, CD16 is used to identify active NK cells in immunohistochemical studies, while CD57 is expressed on NK cell-like elements. The aim of this study was to evaluate tissue presence of NK cells in a series of colorectal adenocarcinomas and to attempt to correlate their presence with clinical and pathological variables and prognostic markers of colorectal cancer. Methods From June 1997 to May 2000, 82 patients from our Department underwent colectomy, due to a diagnosis of colorectal adenocarcinoma of conventional histologic type. NK cell presence was examined via standard 3-step immunohistochemical analysis (ABComplex), which was performed on formalin-fixed, paraffin-embedded tissue sections. Briefly, two appropriate monoclonal antibodies were used (anti-Fc Gamma Receptor II, CD16 and an equivalent to Leu-7, specific for CD-57, Dako, Glostrup, Denmark) at dilutions of 1/200 with overnight incubation. Antigen retrieval was necessary and was performed by usual microwave treatment. In the examined samples, tissue identification of NK cells was based on strong CD16 immunostaining, which was frequently accompanied by CD57 immunoreactivity. Tissue sections from hypertrophic tonsils were used as positive markers. Diaminobenzidine tetrahydrochloride 0.06% in phosphate-buffered saline buffer containing 0.03% hydrogen peroxide was used as a chromogen. Images were acquired using a Zeiss Axiolab microscope (Carl Zeiss GmbH, Jena, Germany) with a mechanical stage, fitted in a Sony-iris CCD video camera (Sony, Tokyo, Japan). The video camera was connected to a Pentium II PC, loaded with the appropriate image analysis software (Sigma Scan Pro, Science, Erkrath, Germany). Slides were examined at a ×200 magnification. The ratio, expressed in percentile proportion (%), between the number of immunohistochemically positive-stained cells and the total number (stained and unstained) of lymphocytes was calculated. All tumors had been characterized according to the following classical clinical and pathological variables: patient gender (male 52, female 30, 63.4% and 36.6%, respectively) and age (mean: 75.9, median: 76, ranging between 35–95 years), tumor location (rectum 27, 32.9%, sigmoid 26, 31.7%, descending colon 4, 4.8%, transverse colon 5, 6%, ascending colon 10, 12.1%, cecum 10, 12.1%), size (mean diameter: 4.4 cm, median diameter: 4 cm, ranging between 1.2–10 cm), grade (I: 24 cases, 29.3%, II: 48 cases, 58.5%, III: 10 cases, 12.2%), bowel wall invasion (present in 66 cases, 80.5%), lymph node metastases (present in 33 cases, 40.2%) and Dukes' stage (A: 5, 6.1%, B: 44, 53.7%, C: 29, 35.4%, "D": 4, 4.9%). All "D" Dukes' stage cases involved hepatic metastases. The tumors were categorized according to their location into two larger groups: those involving the rectum as well as the left colon (57 cases, 69.5%) and those involving the right colon (25 cases, 30.5%). This was performed for reasons of statistical analysis. Moreover, positive cases were divided into two groups: those in which immunopositive cells ranged from 0–9% of the total number of lymphocytes ("weak" tissue presence of NK cells) and those in which they were ≥10% ("strong" NK cell presence). This was performed somewhat arbitrarily, as we considered that the cutoff level of 10% is in accordance to what a pathologist would consider to be the cutoff level between "negative" and "positive" cases in a qualitative examination, and in order to perform statistical analysis. Associations of NK cell presence with the patients' sex, tumor location, grade, and presence of bowel wall invasion, as well as lymph node metastases and Dukes' stage were examined using chi-square statistics; associations with the patients' age and tumor size were determined using the Mann-Whintey U test. Results NK cells were detectable in the primary tumor site of 79 cases (96.3%); their percentages were up to 32% of the primary site lymphocytes (Figure). Of the 33 cases with metastatic lymph nodes, NK cells were found in 27 cases (81.8%); moreover, NK cells were found among liver lymphocytes in 3 of the total 4 cases with hepatic metastases (75% of stage "D" cases). Figure 1 Presence of NK cells within cancerous formations. (ABC, magnification ×250). Forty-nine of the 82 primary tumors (59.8%) had a "weak" tissue presence of NK cells (0–9%), whereas the remaining 33 primary tumors (40.2%) had a "strong" NK cell presence (≥10%). Of the 33 cases with metastatic lymph nodes, 18 cases (54.5%) had "weak" and 15 "strong" NK cell presence. NK cell presence in the primary tumor site was not associated with the patients' sex (chi-square, p = 0.462), tumor size (Mann-Whintey U test, p = 0.377), tumor location (chi-square, p = 0.262), tumor differentiation (chi-square, p = 0.556), wall invasion (chi-square, p = 0.999), the presence of metastatic lymph nodes (chi-square, p = 0.720), or Dukes' stage (chi-square, p = 0.992). However, a negative association between the presence of NK cells at the primary tumor site with the patients' age was noticed (Mann-Whitney U test, p = 0.031). Moreover, the presence of NK cells in the metastatic lymph nodes was not associated with the patients' sex (chi-square, p = 0.999), age (Mann-Whintey U test, p = p = 0.212), tumor size (Mann-Whintey U test, p = 0.729), tumor location (chi-square, p = 0.442), tumor differentiation (chi-square, p = 0.872), wall invasion (chi-square, p = 0.999), or Dukes' stage (C or D) (chi-square, p = 0.308). Discussion Although colorectal adenocarcinoma has been characterized as a model neoplasm in which clinical prognosis is mainly determined by the histological stage, pathologic markers often fail to predict the prognosis of a great number of patients. This has necessitated the evaluation of several alternative prognostic markers [ 1 ]. Immunohistochemical studies have proved useful in the evaluation of such potential markers; although they are relatively cheap and easy-to-perform, they may be as effective as other advanced molecular techniques in determining the role of several molecules in human carcinogenesis [ 6 ]. CD16 and CD57 were chosen in the present study, as they are markers of the presence of NK cells, a cellular line which could reasonably play a significant role in the pathophysiology of colorectal adenocarcinoma. NK cells were considerably present in the primary tumor site, as well as the metastatic lymph nodes of many cases in our series. This observation is in line with data deriving from the literature [ 7 ]. NK cell presence had no correlation with the clinical or pathological variables of our series, besides a negative association between their presence at the primary tumor site and the patients' age (p = 0.031), i.e. less NK cells were found in the stroma of the primary tumor site in the older patients of our study. Reports concerning alterations of the number and/or the cytotoxicity of NK cells in patients of older ages seem to be controversial [ 8 - 14 ]; this has been attributed to the diversity of the parameters examined in these studies [ 8 ]. In most reports, advanced age results in an increase of the number of circulating NK cells [ 8 - 10 ]; however, other studies point out that although total levels of NK cells actually remain steady, some of their biologically active sub-populations actually diminish [ 11 , 12 ]. The cytotoxicity of NK cells on the other hand, has been reported to increase [ 13 ], remain steady [ 8 , 14 ] or diminish [ 12 ] in patients of older age; some reports connect presence of certain nutrients and hormonal factors with the maintenance of NK cytotoxicity in these patients [ 8 , 14 ]. However, what seems to be pivotal for rapid and efficient migration of NK cells from the circulation to the tumor stroma, is the expression of appropriate adhesion molecules [ 15 ]. As expression of adhesion molecules in lymphocytes, monocytes and the interstitial tissue decreases with age [ 16 ] this might result in decreased adhesion molecule-mediated migration of NK cells to the tumor stroma in the older-aged patients of our series; this could possibly explain the negative association observed in our study [ 5 , 15 ]. In conclusion, our findings suggest that less NK cells are found in the stroma surrounding the primary tumor site in older patients with colorectal adenocarcinoma. This could possibly be attributed to decreased adhesion molecule-mediated migration; however, this hypothesis needs to be further investigated through more studies. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC517933.xml
524484	Evolutionary relationships of Aurora kinases: Implications for model organism studies and the development of anti-cancer drugs	Background As key regulators of mitotic chromosome segregation, the Aurora family of serine/threonine kinases play an important role in cell division. Abnormalities in Aurora kinases have been strongly linked with cancer, which has lead to the recent development of new classes of anti-cancer drugs that specifically target the ATP-binding domain of these kinases. From an evolutionary perspective, the species distribution of the Aurora kinase family is complex. Mammals uniquely have three Aurora kinases, Aurora-A, Aurora-B, and Aurora-C, while for other metazoans, including the frog, fruitfly and nematode, only Aurora-A and Aurora-B kinases are known. The fungi have a single Aurora-like homolog. Based on the tacit assumption of orthology to human counterparts, model organism studies have been central to the functional characterization of Aurora kinases. However, the ortholog and paralog relationships of these kinases across various species have not been rigorously examined. Here, we present comprehensive evolutionary analyses of the Aurora kinase family. Results Phylogenetic trees suggest that all three vertebrate Auroras evolved from a single urochordate ancestor. Specifically, Aurora-A is an orthologous lineage in cold-blooded vertebrates and mammals, while structurally similar Aurora-B and Aurora-C evolved more recently in mammals from a duplication of an ancestral Aurora-B/C gene found in cold-blooded vertebrates. All so-called Aurora-A and Aurora-B kinases of non-chordates are ancestral to the clade of chordate Auroras and, therefore, are not strictly orthologous to vertebrate counterparts. Comparisons of human Aurora-B and Aurora-C sequences to the resolved 3D structure of human Aurora-A lends further support to the evolutionary scenario that vertebrate Aurora-B and Aurora-C are closely related paralogs. Of the 26 residues lining the ATP-binding active site, only three were variant and all were specific to Aurora-A. Conclusions In this study, we found that invertebrate Aurora-A and Aurora-B kinases are highly divergent protein families from their chordate counterparts. Furthermore, while the Aurora-A family is ubiquitous among all vertebrates, the Aurora-B and Aurora-C families in humans arose from a gene duplication event in mammals. These findings show the importance of understanding evolutionary relationships in the interpretation and transference of knowledge from studies of model organism systems to human cellular biology. In addition, given the important role of Aurora kinases in cancer, evolutionary analysis and comparisons of ATP-binding domains suggest a rationale for designing dual action anti-tumor drugs that inhibit both Aurora-B and Aurora-C kinases.	Background The Auroras are a conserved family of serine/threonine kinases which have essential functions in cell division [ 1 , 2 ]. In mitosis, Aurora kinases are required for chromosome segregation, condensation and orientation in the metaphase plate, spindle assembly, and the completion of cytokinesis. Model organism studies have played a pivotal role in functional characterization of Aurora kinases. Aurora kinases were first identified as mutant alleles in Drosophila melanogaster (fruitfly) that caused defective spindle-pole formation [ 3 ]. Subsequently, Drosophila was found to have a second Aurora homolog [ 4 ], and the nematode, Caenorhabditis elegans , similarly has two Aurora-like genes [ 5 , 6 ]. The fungi, Saccharomyces cerevisiae and Schizosaccharomyces pombe , have a single Aurora, known as increase-in-ploidy 1 (Ipl1) [ 7 ] and Aurora-related kinase 1 (Ark1) [ 8 ], respectively. Among cold-blooded vertebrates, Aurora kinases have been most widely studied in the frog, Xenopus laevis , which has two kinases; Aurora-A and Aurora-B [ 9 , 10 ]. More recently discovered is a third Aurora kinase called Aurora-C in rodents and humans [ 11 ]. The Aurora kinases are mitotic kinases that generally associate with chromosomes, often in complexes with other proteins, and interact with cytoskeletal components in cell division. The three mammalian Aurora kinases appear at specific locations during mitosis. Aurora-A, the "polar kinase", primarily associates with the separating centrosomes while Aurora-B, the "equatorial kinase", is a chromosomal passenger protein [ 1 ]. The least studied Aurora kinase, Aurora-C, appears to be localized to the centrosome from anaphase to telophase and is highly expressed in the testis [ 11 , 12 ]. Recent studies indicate that all three Aurora kinases have strong associations with cancer. Aurora-A has been mapped to a region in the human chromosome (20q13.2-13.3) that is amplified in cancer cell lines and primary tumors [ 13 , 14 ]. Transfected mouse cell lines with Aurora-A have been shown to cause tumors when injected into nude mice [ 14 , 15 ] and a polymorphic variant (amino acid substitution Phe31Ile) has been associated with human colon tumors [ 16 ]. Expression levels of Aurora-B [ 17 ] and Aurora-C [ 12 ] were elevated in several cancer cell lines relative to normal fibroblasts. Aurora-C is located on chromosome 19q13.2 to 13.4, a region associated with loss of heterozygosity in ovarian cancer [ 18 ] and pancreatic carcinomas [ 19 ]. Thus, the inhibition of one or more Aurora kinases might be a novel chemotherapeutic strategy against cancer [ 20 ]. Recently, several reports by research groups in pharmaceutical and biotechnology companies describe small molecules that target the ATP-binding domain of Aurora kinases, and have effects in human tumor cell lines [ 21 - 23 ]. Despite the importance of model organisms in understanding Aurora kinase function, the evolutionary relationships among these variants are unclear. Two previous phylogenetic analyses of Aurora kinases were incomplete because the contemporary complement of Aurora kinases was unavailable [ 4 ] or certain family members, namely the Aurora-C kinases, were excluded [ 1 ]. Here, we present an evolutionary analysis of all known Aurora kinases. We show that vertebrate Aurora kinases evolved through a series of gene duplication events from a chordate ancestor, and that they are highly distinct from invertebrate homologs. Moreover, the recent divergence, thus high level of sequence similarity, of human Aurora-B and Aurora-C suggests a novel anti-cancer strategy which might simultaneously target the ATP-binding domains of this kinase pair with dual action inhibitors. Results and discussion Aurora Evolution in Chordates In order to construct a comprehensive phylogenetic tree, GenBank was searched for all possible Aurora kinases. In addition to previously published Aurora kinase sequences, further chordate and urochordate Aurora homologs were found by using mammalian Aurora-A, Aurora-B and Aurora-C protein sequences as queries in BLASTP or TBLASTN [ 24 ] searches of the genomes of the pufferfish, Takifugu rubripes [ 25 ], the zebrafish, Danio rerio [ 26 ], and the ascidian, Ciona intestinalis [ 27 ]. Multiple sequence alignments show that the Aurora kinase family is highly conserved among species (Fig. 1 ). Pairwise sequence comparisons estimate that the mean proportion of similar amino acids (based on the Blosum62 matrix) is much higher among all the different families of Aurora-A, Aurora-B and Aurora-C of vertebrates (0.84 ± 0.5) than within the same family (Aurora-A or Aurora-B) between vertebrates and invertebrates species (0.69 ± 0.3 for both families). This would suggest a recent evolutionary radiation of Aurora families within vertebrates. Figure 1 Multiple sequence alignment of representative Aurora-A (AurA), Aurora-B (AurB), and Aurora-C (AurC) kinases, and their homologs (Air1, Air2, ARK1 and Ipl1). N-terminal regions which are species-specific and could not be accurately aligned are excluded, although the numbering of residues begins at the starting amino acid for that particular peptide. Progressive darker shading indicates conservation of amino acid residues in 60%, 80% and 100% of the sequences, respectively. Dark line at the top of the sequence blocks indicates those regions used in the phylogenetic analyses (Also see additional file 1 and 2 ). Species include Homo sapiens (hosa), Mus musculus (mus), Danio rerio (dare), Takifugu rubripes (taru), Xenopus laevis (xela), Ciona intestinalis (ciin), Drosophila melanogaster (drme), Caenorhabditis elegans (cael), Saccharomyces cerevisiae (sace) and Schizosaccharomyces pombe (scpo). The program CLUSTALW [41] was used to constructed the initial alignment which was subsequently refined manually. Phylogenetic trees constructed using four methodologies, all rooted using polo-like kinases type 4 (PLK4), show that all vertebrate Auroras form a clade distinct from those of invertebrates (Fig. 2 ). The phylogenetic tree constructed by the neighbour-joining distance method shows moderate boot-strap support (67%) for the evolution of all vertebrate Auroras from a urochordate ancestor, represented by the ascidian, C. intestinalis . The use of alternative kinase families, other than PLK4, to root the tree did not alter the internal topology of the Aurora clade. Although its genome sequence is incomplete, C. intestinalis likely has only a single Aurora homolog since other probable kinase open reading frames associated with the next top five BLASTP [ 24 ] hits did not cluster with Auroras from other species in phylogenetic trees. Figure 2 Phylogenetic tree of Aurora-A, Aurora-B, and Aurora-C kinases rooted by PLK4 kinases. Major organism groups (with colours, fonts) are mammals (red, bold italic), cold-blooded vertebrates (deep blue, italic), urochordates (orange, italic), invertebrates, (purple, italic), plants (green, italic), fungi (black, italic) and protists (light blue, italic). "Original" indicates the first Aurora identified from Drosophila melanogaster [3]. Plant sequences are identified by their Genbank accession number. Stacks of numbers show, in descending order, the percent occurrence of nodes in greater than 50% of 1000 bootstrap replicates of neighbor joining (plain text) and maximum parsimony (italicized text) analyses or greater than 50% of 10000 quartet puzzling steps of maximum likelihood analysis (in curved parentheses) or Bayesian posterior probability (only 0.90 or greater, in square parentheses). Asterisks ("*") indicate those nodes supported 70% or greater by the first three tree-building methods and 0.90 Bayesian posterior probability. Nodes with one or two values less than 50% have dashes ("-") while values less than 50% are unmarked. Scale bar represents 0.1 expected amino acid residue substitutions per site. Among true vertebrates, our phylogenetic tree shows that the Aurora kinases underwent two major gene duplication events. The first split in cold-blooded vertebrates lead to the formation of two Aurora subfamilies. One branch encompasses all known vertebrate Aurora-A sequences in a single orthologous lineage that includes fishes, amphibians and mammals. This family includes previously identified Aurora-A kinases in Xenopus laevis , rodents and humans as well as a new putative ortholog in T. rubripes . The second family, previously known as Aurora-B [ 2 , 28 ] consists of cold-blooded vertebrate and mammalian Aurora-B as well as mammalian Aurora-C. Mammalian Aurora-B and Aurora-C are similarly related to the cold-blooded vertebrate Aurora presently known as "Aurora-B" in amphibians ( X. laevis ) and fish ( D. rerio and T. rubripes ). Searches of T. rubripes and D. rerio protein and DNA sequence databases detected several other putative serine/threonine kinase homologs but none were Auroras according to phylogenetic analyses. Thus, cold-blooded vertebrates appear to have only a single Aurora-A ortholog, a single Aurora-B-like homolog, and lack an Aurora-C ortholog. Conversely, Aurora-A, Aurora-B and Aurora-C appear to be ubiquitous to mammals (at least placentals) where they are encoded by separate chromosomal loci. It would appear that mammalian Aurora-B and Aurora-C evolved from a duplication event involving the ancestral Aurora-B found in cold-blooded vertebrates. This depiction of the evolutionary relationships of vertebrate Auroras was consistently determined by four different phylogenetic methods with high bootstrap or Bayesian posterior probability values (Fig. 2 ). Comparisons of human Aurora-B and Aurora-C sequences to the resolved 3D structure of human Aurora-A [ 29 ] lends further support to the evolutionary scenario that vertebrate Aurora-B and Aurora-C are closely related paralogs (Fig. 3a ). Of the 26 residues lining the ATP-binding active site, only three vary among the different human Aurora kinases; Leu215, Thr217 and R220 (numbering and residue identity based on Aurora-A), and all of these variants were specific to Aurora-A (Fig. 3b ). Aurora-B and Aurora-C did not vary in their active site residues. Furthermore, all three Auroras have a carboxy-terminal destruction box (D-box) but only Aurora-A has the necessary amino-terminal A-box (also known as the D-box activating-domain) for its functional activation [ 30 , 31 ]. Collectively, these comparisons of structure and motifs support the phylogeny depicting an early divergence of Aurora-A from an Aurora-B / Aurora-C clade. Figure 3 Comparisons of the catalytic domains of human Aurora-A, Aurora-B and Aurora-C kinases. A. Crystal structure of the catalytic domain of Human Aurora kinase with an adenosine molecule shown in the binding pocket (PDB ID 1muoA) [29]. Residues lining the active site are colored purple when invariant and red when variant. B , Multiple sequence alignment of Auroras. Using the same color scheme as the structure in panel A, residues identified to be lining the active site are identified with invariant residues among all three Auroras marked with an asterisk. Of the 26 residues lining the active site, only three vary among the different human Aurora kinases; Leu215, Thr217 and R220 (numbering and residue identity based on Aurora-A), and all of this variation was found in Aurora-A. Non-chordate Evolution The Aurora kinases of plants and invertebrates are all outgroup lineages to chordates / urochordates (Fig. 2 ). Although all phylogenetic methods strongly support the monophyly of chordate Aurora kinases, the exact ordering among nodes leading to the various plant and invertebrate clusters were not resolved with similarly high bootstrap or probability values. Placement of plant Aurora kinases between chordates and invertebrates might be an artifact of tree construction methods. (Plant, protist, fungal and invertebrate lineages were all highly diverged from vertebrate Aurora kinases as witnessed by their longer branch lengths.) The earliest lineages of the Aurora tree are those fungal model organisms with a single Aurora-like homolog S. cerevisiae (Ipl1) and S. pombe (Ark1). Other basal branches are the amitochondrial fungi, Encephalitozoon cuniculi , and the kinetoplast protist, Leishmania major [ 32 ]. Invertebrate Aurora kinases, including those of the model organisms C. elegans and D. melanogaster , occupy separate early branches and are not, as their current names suggest, orthologs to either Aurora-A or Aurora-B of vertebrates. An unrooted phylogenetic tree with only model organism species shows the same topology of vertebrate Auroras as the more species-rich tree rooted by PLK4 kinases (Fig. 4 ). However, similar kinases from C. elegans and D. melanogaster now cluster together. The unrooted tree suggests that the invertebrate Aurora-B kinase family evolved prior to the invertebrate Aurora-A kinase family although further examples from other species are desirable to confirm this hypothesis. The consensus scenario in both rooted and unrooted trees is that vertebrate Aurora kinases are paralogous, rather than orthologous, to their invertebrate counterparts. Figure 4 Unrooted phylogenetic tree of Aurora kinases from human and model organisms. Tree was constructed using the maximum likelihood quartet puzzling method [43]. Scale bar represents 0.1 expected amino acid residue substitutions per site. Confidence estimates of nodes, fonts, and colours of species names correspond to Fig. 2. Model Organisms in Context Aurora-B and Aurora-C, as specific innovations in mammals, might have distinct protein-binding partners and cellular functions from those of Aurora-B kinases in amphibians. The perturbation of Aurora-B function in different systems suggests variable kinetochore-microtubule interactions [ 33 ]. Transfection of normal rat kidney cells with a kinase-inactive, dominant negative form of Aurora-B caused multiple defects in mitosis [ 34 ] while an Aurora-B kinase inactivating antibody seemed to have milder effects in Xenopus tissue culture cells [ 35 ]. Xenopus Aurora-A functions in the extrusion of the first polar body [ 36 ] while in C. elegans Aurora B plays a similar role [ 5 ]. Also, C. elegans Aurora-B binds to a protein CSC-1 which has no homolog in other studied systems [ 37 ]. While these studies used different experimental methods, the lack of direct orthology among vertebrate and invertebrate Aurora-A and Aurora-B might also account for functional differences in these systems. The evolutionary analysis presented here also suggests revisiting the present Aurora nomenclature. Adams et al. [ 28 ] proposed a naming scheme where, irrespective of species, the original Aurora is known as Aurora-A (also called AIRK1, Aurora, Aurora-2, AIK, BTAK, human STK15, mouse STK6 and others), followed by Aurora-B (also known as AIRK-2, IAL, Aurora-1, AIK2, STK12 and others) and Aurora-C (or STK13). However, the proposed nomenclature fails to reflect evolutionary, and possibly functional, relationships among the Auroras. We suggest that Aurora-A be retained as the name for all orthologs in mammals and cold-blooded vertebrates. While Aurora-B and Aurora-C seem appropriate for mammalian versions, the ancestral cold-blooded vertebrate "Aurora-B" might be renamed "Aurora-BC". As for invertebrates, the so-called Aurora-A or Aurora-B genes are clearly not orthologs to their respective vertebrate counterparts. However, introducing a new nomenclature here might simply add further confusion to the field. Evolution of an Anti-Cancer Target There have been several recent reports of Aurora kinase inhibitors that are under development by pharmaceutical or biotechnology companies for cancer treatment. The compounds Hesperadin (Boehringer Ingelheim [ 21 ]) and ZM447439 (AstraZeneca [ 22 ]) are suggested to be targeted to Aurora-B. While both studies show lesser levels of compound inhibition of Aurora-A as well as several other kinases, neither report included Aurora-C in their kinase profile. Selective inactivation of multiple kinases is not an undesirable pharmaceutical profile for a small molecule inhibitor and, in fact, could be the best strategy to achieve maximal clinical efficacy of an anti-cancer agent [ 38 ]. Indeed, an intense area of anti-cancer research is the development of small molecular ATP analogues that generally target the kinase domain of protein kinases [ 39 ]. For example, Gleevec (also known as imatinib and made by Novartis) for chronic myelogenous leukemia, is a small-molecule inhibitor that targets BCR-ABL, c-Kit and platelet-derived growth factor receptor kinases [ 40 ]. Recently, a selective inhibitor of all three Aurora kinases, VX-680 (made by Vertex Pharmaceuticals), was reported to inhibit cell-cycle progression and induce apoptosis in various human tumor cell types and in vivo xenograft models [ 23 ]. Interestingly, although VX-680 is a potent inhibitor of all three Aurora kinases, its apparent inhibition constant is much lower for Aurora-A (0.6 nM) than for either Aurora-B (18 nM) or Aurora-C (4.6 nM). Again, the compound's greater affinity for Aurora-A, relative to Aurora-B and Aurora-C is compatible with the proposed evolutionary scenario of mammalian Auroras. Conclusions Evolutionary analysis shows that cell division Aurora kinases, while consistent in theme throughout eukaryotes, have undergone lineage-specific expansions and specialization in metazoans. Aurora-C is the least known of the Aurora kinases. Yet as an evolutionary innovation in mammals, further studies are very much warranted from the perspectives of better understanding its potential roles in both cell replication and tumor progression. A better functional understanding of Aurora-C would help clarify the evolutionary relationships of Aurora-B and Aurora-C in mammals relative to the ancestral Aurora-BC in cold-blooded vertebrates. Additionally, the close evolutionary and structural relationships between mammalian Aurora-B and Aurora-C offers the tantalizing opportunity to design dual kinase inhibitors that might circumvent potential tumor cell resistance to mono-target chemotherapeutics. Methods Database searches All Aurora kinase orthologs and paralogs were initially collected from GenBank nonredundant protein database by performing separate searches using BLASTP [ 24 ] with human Aurora-A, Aurora-B and Aurora-C proteins as query sequences and a cut-off E-value of 1.0e-10. Since this dataset included additional kinases to the Auroras, preliminary multiple sequence alignments and phylogenetic analysis using program CLUSTALW v1.7 [ 41 ] served to identify the clade of all known Aurora kinases. Takifugu rubripes , Danio rerios and Ciona intestinalis homologs were obtained by BLASTP and TBLASTN [ 24 ] of species-specific protein and DNA sequence databases, respectively. The top five homologs from each species retrieved from separate searches with human Aurora-A, Aurora-B and Aurora-C were entered into a preliminary phylogenetic analysis using all retrieved Aurora kinases from Genbank. These analyses revealed that T. rubripes and D. rerios had orthologs to X. laevis Aurora-A and Aurora-B but not mammalian Aurora-C. C. intestinalis had a single Aurora-like kinase. Phylogenetic and structure analysis PLK4 kinases were selected as the outgroup for phylogenetic analyses because they were the most similar non-Aurora kinases to either human Aurora-A, Aurora-B or Aurora-C in multiple BLASTP [ 24 ] searches of the non-redundant protein database of GenBank. Using alternative kinases as outgroups made no difference to the topology of the Aurora clade. Initial multiple sequence alignments were performed using the program CLUSTALW v1.7 [ 41 ] with default settings and subsequently, refined manually using the program SEQLAB of the GCG Wisconsin Package v11.0 software package (Accelrys, San Diego, CA, USA). We removed regions with residues that could not be unambiguously aligned or that contained insertions or deletions. The final multiple sequence alignment was 240 amino acids in length. Pairwise comparisons for the proportion of similar residues were estimated from the length of the shortest sequence without gaps and the Blosum62 weighting matrix as implemented in the program OLDDISTANCES in GCG. We constructed phylogenetic trees using distance neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood quartet puzzling (QP), and Bayesian posterior probabilities (BP). NJ trees were based on pair wise distances between amino acid sequences using the programs NEIGHBOR and PROTDIST (Dayhoff option) of the PHYLIP 3.6 package [ 42 ]. The programs SEQBOOT and CONSENSE were used to estimate the confidence limits of branching points from 1000 bootstrap replications. ML tree topologies were constructed using the software PUZZLE 4.0 [ 43 ], employing 1000 puzzling steps, the JTT substitution matrix, estimation of rate heterogeneity using the gamma distribution model with eight rate categories, and the gamma-parameter estimation from the dataset. MP analysis was performed using PAUP4.0b5 software [ 44 ] where the number and lengths of minimal trees were estimated from 100 random sequence additions, while confidence limits of branch points were estimated by 1000 bootstrap replications. BP trees were constructed using the software MrBayes v3.0B4 [ 45 , 46 ]. Bayesian analysis used the mixed model of sequence evolution with random starting trees. Markov chains were run for 10 6 generations, burn-in values were set for 10 4 generations, and trees sampled every 100 generations. All trees were visualized using the program TREEVIEW v1.6.6 [ 47 ]. For the Aurora kinase phylogeny rooted with PLK4 kinases shown in Fig. 2 , the log likelihood of the final ML tree was -8059.78. Four minimal length MP trees were recovered, 1522 steps in length with a consistency index (CI) of 0.5802 and a retention index (RI) of 0.6016. The variable branch arrangements were terminal nodes (human, pig and cow Aurora-B) which did not affect the central findings. For the unrooted phylogeny of Aurora kinases of model organisms shown in Fig. 3 , the log likelihood of the final ML tree was -4469.20. A single minimal length MP trees were recovered, 779 steps in length with a consistency index (CI) of 0.7214 and a retention index (RI) of 0.2786. The SwissPDBviewer program [ 48 ] was used to obtain the surface representation of human Aurora-A kinase (PDB ID 1muoA). The active site residues, defined as being within 5A of the ADP cofactor, were identified using the program CAST [ 49 ]. The multiple sequence alignment for the three human Aurora kinase proteins was obtained using CLUSTALW [ 41 ]. Multiple sequence alignment and sequence GenBank accession numbers are available as Supplementary Information [see additional file 1 and 2 ]. Authors' contributions JRB conceived the study, performed the phylogenetic analysis and drafted the manuscript. KKK performed the 3D structural analyses and contributed to the draft manuscript. MLB participated in the preliminary phylogenetic analysis and contributed to the draft manuscript. PS and DRP both contributed to the draft of the manuscript and added key references. Supplementary Material Additional File 1 Multiple sequence alignment of edited Aurora and Plk4 kinases used to produce the phylogeny shown in Fig. 2 . Click here for file Additional File 2 Accession numbers (gi), Locus link Ids (LID) or genome predicted protein Ids for Aurora and PLK kinases as labelled in the Supplementary Data multiple sequence alignment file S1. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524484.xml
545052	Phase I vaccination trial of SYT-SSX junction peptide in patients with disseminated synovial sarcoma	Background Synovial sarcoma is a high-grade malignant tumor of soft tissue, characterized by the specific chromosomal translocation t(X;18), and its resultant SYT-SSX fusion gene. Despite intensive multimodality therapy, the majority of metastatic or relapsed diseases still remain incurable, thus suggesting a need for new therapeutic options. We previously demonstrated the antigenicity of SYT-SSX gene-derived peptides by in vitro analyses. The present study was designed to evaluate in vivo immunological property of a SYT-SSX junction peptide in selected patients with synovial sarcoma. Methods A 9-mer peptide (SYT-SSX B: GYDQIMPKK) spanning the SYT-SSX fusion region was synthesized. Eligible patients were those (i) who have histologically and genetically confirmed, unresectable synovial sarcoma (SYT-SSX1 or SYT-SSX2 positive), (ii) HLA-A*2402 positive, (iii) between 20 and 70 years old, (iv) ECOG performance status between 0 and 3, and (v) who gave informed consent. Vaccinations with SYT-SSX B peptide (0.1 mg or 1.0 mg) were given subcutaneously six times at 14-day intervals. These patients were evaluated for DTH skin test, adverse events, tumor size, tetramer staining, and peptide-specific CTL induction. Results A total of 16 vaccinations were carried out in six patients. The results were (i) no serious adverse effects or DTH reactions, (ii) suppression of tumor progression in one patient, (iii) increases in the frequency of peptide-specific CTLs in three patients and a decrease in one patient, and (iv) successful induction of peptide-specific CTLs from four patients. Conclusions Our findings indicate the safety of the SYT-SSX junction peptide in the use of vaccination and also give support to the property of the peptide to evoke in vivo immunological responses. Modification of both the peptide itself and the related protocol is required to further improve the therapeutic efficacy.	Background Synovial sarcoma is a relatively rare, high-grade malignant tumor of soft tissue, characterized by biphasic or monophasic histology, specific chromosomal translocation t(X;18), and its resultant SYT-SSX fusion gene[ 1 ]. This tumor affects mostly adolescents and young adults. The 5-year survival rates of patients with this localized disease have ranged from 66% to 80% in the current literature [ 2 - 5 ]. However, the majority of metastatic or relapsed diseases still remain incurable despite intensive multimodality therapy. Therefore there is a need for additional new therapeutic options other than conventional surgery, radiotherapy, and chemotherapy. Vaccination of tumor antigenic peptide serves as a commonly accepted method in anti-cancer immunotherapy[ 6 , 7 ]. This is based on the rationale that T cells recognize antigenic peptide in the context of MHC molecules on the tumor cell or antigen presenting cells through the T cell receptor, which elicits subsequent anti-tumor immune responses. Identification of antigenic peptides recognized T cells enabled us to apply vaccination trials to a variety of tumors, except bone and soft tissue sarcomas[ 8 ]. Currently tumor specific chromosomal translocations are defined in leukemias, lymphomas, and sarcomas[ 9 , 10 ]. The fusion regions of translocation products are specifically expressed by its corresponding tumors, thereby serving as targets of great potential for tumor specific therapies, including immunotherapy[ 11 , 12 ]. We[ 13 , 14 ] found that SYT-SSX fusion gene-derived peptides can be recognized by circulating CD8+ T cells in patients with synovial sarcoma and elicit HLA-restricted, tumor specific cytotoxic responses by in vitro stimulations. In this study, we conducted a phase I pilot trial of vaccination of a SYT-SSX-derived junction peptide in elected synovial sarcoma patients. Methods Peptide A 9-mer peptide (SYT-SSX B: GYDQIMPKK) spanning the SYT-SSX fusion region was synthesized under good manufacturing practice (GMP) conditions by Multiple Peptide Systems (San Diego, CA). The identity of the peptide was confirmed by mass spectral analysis, and was shown to have more than 98% purity when assessed by high pressure liquid chromatography analysis. The peptide was delivered us in the form of a freeze-dried, sterile white powder. It was dissolved in 1.0 ml of physiological saline (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) and stored at -80°C until just before usage. The affinity of the peptide to HLA-A24 molecules and its antigenicity were determined in previous studies[ 13 , 14 ]. Eligibility The study protocol was approved by the Clinical Institutional Ethical Review Board of the Medical Institute of Bioregulation, Sapporo Medical University, Japan. Eligible patients were those (i) who have histologically and genetically confirmed, unresectable synovial sarcoma (SYT-SSX1 or SYT-SSX2 positive), (ii) HLA-A2402 positive, (iii) between 20 and 70 years old, (iv) ECOG performance status between 0 and 3, and (v) who gave informed consent. Exclusion criteria included (i) prior chemotherapy, steroid therapy, or other immunotherapy within the past 4 weeks, (ii) presence of other cancers that might influence the prognosis, (iii) immunodeficiency or a history of splenectomy, (iv) severe cardiac insufficiency, acute infection, or hematopoietic failure, (v) ongoing breast-feeding, (vi) unsuitability for the trial based on the clinical judgment of the doctors involved. This study was carried out at the Department of Orthopaedic Surgery, Sapporo Medical University Hospital from June 2003 until the end of September 2004. Vaccination schedule Vaccinations with SYT-SSX B peptide were administered subcutaneously into the upper arm six times at 14-day intervals. In order to set up a dose-escalation trial, the patients were separated into the two groups. Each group included three patients. Those from group 1 received 0.1 mg and group 2 participants received 1.0 mg. Delayed-type hypersensitivity (DTH) skin test Delayed-type hypersensitivity (DTH) skin test was performed at each vaccination. The peptide (10 μg) solution in physiological saline (0.1 ml) or physiological saline alone (0.1 ml) were separately injected intradermally into the forearm. A positive reaction was defined as a diameter of erythema of more than 4 mm, 48 hr after the injection. Toxicity evaluation Patients were examined closely for signs of toxicity during and after vaccination. Adverse events were recorded using the National Cancer Institute Common Toxicity Criteria (NCI-CTC). Clinical response evaluation Physical examinations and hematological examinations were monitored before and after each vaccination. Tumor size was evaluated by computed tomography (CT) scans before treatment, and again after three vaccinations, and then at the end of the study period. A complete response (CR) was defined as complete disappearance of all measurable diseases. A partial response (PR) was defined as a >= 50% decrease from the baseline in size of all measurable lesions (sum of products of maximal perpendicular diameters) lasting for a period of at least 4 weeks. Progressive disease (PD) was defined as an increase in the sum of the bi-dimensional measurements of all known disease sites by at least 25% or by the appearance of new lesions. No change (NC) was defined as the absence of matched criteria for CR, PR, or PD. Tetramer staining HLA-A24/peptide tetramers (HLA-A24/B, HLA-A24/R49.2, and HLA-A24/HIV) were previously constructed [ 13 - 15 ]. Flowcytometric analysis was performed by taking peripheral blood mononuclear cells (PBMCs) from patients. PBMCs were taken at pre-vaccination and again one week after 1 st , 3 rd , and 6 th vaccination. Cells were stained with PE-labeled tetramers at 37°C for 20 min and a FITC-conjugated anti-CD8 mAb (Becton Dickinson) at 4°C for 30 min. Analysis of stained PBMCs was performed using FACScan (Becton Dickinson) and CellQuest software (Becton Dickinson). The frequency of CTL precursors was calculated as the number of tetramer positive cells / the number of CD8 + cells. CTL induction Cytotoxic T lymphocytes (CTLs) were induced from the PBMCs of patients using SYT-SSX B peptides according to the method described before[ 13 , 14 ]. The cytotoxic activity was evaluated by 6-h 51 Cr release assay[ 13 ]. As target cells, synovial sarcoma cell lines (Fuji, HS-SY-II, and SW982), an erythroleukemia cell line (K562), and a T-B Lymphoblast hybrid transfected with HLA-A2402 (T2-A2402) were used. Fuji and HS-SY-II were both HLA-A24 and SYT-SSX positive lines. SW982 and K562 were both HLA-A24 and SYT-SSX negative lines used as controls. T2-A2402 cells were used to determine peptide-specific cytotoxicity by pulsation with SYT-SSX B or HIV peptide before labeling. The stimulated CD8 + T cells were mixed with the labeled target cells. After a 6-h incubation period at 37°C, the release of the 51 Cr label was measured by collecting the supernatant, followed by quantification in an automated gamma counter. The percentage of specific cytotoxicity was calculated as the percentage of specific 51 Cr release: [(experimental 51 Cr release - spontaneous 51 Cr release) / (maximum 51 Cr release - spontaneous 51 Cr release)] × 100. Maximum 51 Cr release was measured by incubating the labelled target cells with 2% NP-40, instead of the stimulated CD8 + T cells. CTL induction was determined as successful when specific cyotoxicity of 10% or more was achieved on Fuji, HS-SY-II, and SYT-SSX B peptide-pulsed T2-A2402 cells. Results Patient profiles Six patients were enrolled in the study (Table 1 ). There were four men and two women with an average age of 34.7 years old (range 21–69 years). All patients had multiple metastatic lesions of the lung. A six-time vaccination schedule was completed in three patients, while the remaining three discontinued the vaccination regimen because of rapid disease progression. None of the treatment interruptions were due to the adverse effects of the vaccination. Table 1 Profiles of participants and clinical resoponses Patient no. Age Gender Dose of peptide (mg) Number of vaccination Adverse events DTH skin test Evaluation of CT images 1 69 M 0.1 1 - - PD 2 32 M 0.1 3 - - PD 3 21 F 0.1 6 - - PD 4 21 M 1.0 6 - - PD 5 39 F 1.0 6 Fever - NC 6 26 M 1.0 4 - - PD PD: progressive disease, NC: no change Safety and DTH skin test One patient (case 5) experienced slight fever (grade 1) after the first vaccination. No other adverse events were observed during vaccination. DTH skin test was performed at each vaccination and assessed 48 hr later. Reactions were determined as negative in all patients. Clinical response Recognized disease progression occurred in five out of six patients during the vaccination period (Table 1 , Fig. 1 ). In contrast, one patient (case 5) showed no such rapid progression (Table 1 , Fig. 2 ). These patients, except in case 1, had received systemic multidrug chemotherapy from one to four months before enrolling on this study. Figure 1 CT scan image of the lung of case 3 patient. A: Before vaccination (May 15, 2003). B: After the third vaccination (June 18, 2003). Rapid growth of the metastatic tumors and pleural effusion were seen. Figure 2 CT scan image of the lung of case 5 patient. A: Before vaccination (July 8, 2003). B: After the first vaccination (July 22, 2003). C: After the third vaccination (August 19, 2003). D: After the sixth vaccination (September 16, 2003). The metastatic tumors appeared to be dormant after the first vaccination. Tetramer analysis and CTL induction Peptide-specific immunological responses were evaluated in five patients by using HLA-A24/peptide tetramer analysis and in vitro CTL induction. As determined by flowcytometric analysis using HLA/peptide tetramers (Table 2 ), frequencies of CTLs specific for SYT-SSX B peptide were shown to be at background levels (less than 0.1%) in three patients prior to vaccination. Those frequencies increased after the first (cases 4 and 6) and the third vaccination (case 2) (Fig. 3 ). In the remaining two patients (cases 3 and 5), SYT-SSX B peptide-specific CTLs existed beyond the background levels before vaccination. Of these, B peptide-specific CTL frequencies increased slightly in case 2 upon a series of vaccinations. On the contrary, the CTL frequencies in peripheral blood decreased to the background level after the third vaccination in case 5, whose metastatic diseases remained stable during the vaccination period. For comparison, tetramers with irrelevant peptides were constructed as internal controls and utilized in four patients. Notably, CTL frequencies reacting to those irrelevant tetramers remained under the background level during the course of vaccinations in all patients. Table 2 HLA-A24/peptide tetramer analysis case Pre-vaccination After 1st vac. After 3rd vac. After 6th vac. 2 0.02/N.D 0.02/N.D 3.05 /N.D N.D 3 0.42 /0.02 0.49 /0.02 0.52 /0.02 0.62 /0.01 4 0.06/0.01 0.41 /0.00 0.36 /0.01 0.47 /0.01 5 0.50 /0.06 0.52 /0.01 0.09/0.00 0.03/0.02 6 0.02/0.01 0.15 /0.01 0.08/0.00 N.D N.D: not determined, B peptide tetramer / Control peptide tetramer HLA-A24/R49.2 pepitde tetramer was used as control in the case 3 patient. HLA-A24/HIV pepitde tetramer was used as control in the other patients. Figure 3 Frequency of CTLs analyzed by HLA-A24/peptide tetramers in the case 4 patient. Frequencies of each analysis were described in Table 2. Table 3 depicts the results of CTL induction by in vitro stimulations with SYT-SSX B peptide. Before vaccination, CTLs specific for SYT-SSX B peptide were successfully induced from one patient (case 2) who showed a high frequency of CTL precursors. After the first or third vaccination, CTLs were induced from four of five patients. Figure 4 represents the results of cytotoxicity assay. As shown, CTLs induced from the case 4 patient exhibited cytotoxic activities against T2-A2402 cells pulsed with SYT-SSX B peptide, and synovial sarcoma cell lines expressing HLA-A24 and SYT-SSX (Fuji and HS-SY-II) in various effecter/target ratios examined. In contrast, the cytotoxity was less than 10% against T2-A2402 cells without peptide pulsation, those pulsed with irrelevant HIV peptide, and tumor cells lacking HLA-A24 and SYT-SSX (SW982 and K562). These findings suggest that induction of peptide-specific immune responses in patients with synovial sarcoma, who received the SYT-SSX junction peptide vaccine. Table 3 Induction of peptide specific CTLs case Pre-vaccination After 1st vac. After 3rd vac. After 6th vac. 2 Failure Failure Success N.D 3 Success Success Success Failure 4 Failure Success Success N.D 5 Failure Success Failure Failure 6 Failure Failure Failure N.D N.D: not determined Figure 4 Cytotoxicity of CTLs induced from the case 4 patient. T2-P(-): T2-A2402 cells without peptide pulsation, T2-B: T2-A2402 cells pulsed with SYT-SSX B peptide, T2-HIV: T2-A2402 cells pulsed with HIV peptide. Specific cytotoxicity was observed against T2-A*2402 cells pulsed with SYT-SSX B peptide, and synovial sarcoma cell lines expressing HLA-A24 and SYT-SSX (Fuji and HS-SY-II). E/T: Effecter cells/target cells ratio. Discussion The present study was designed to evaluate the in vivo immunological property of a 9-mer SYT-SSX junction peptide in patients with disseminated synovial sarcoma. A total of 16 vaccinations of the peptide in six patients revealed (i) no serious adverse effects in any case, (ii) suppression of tumor progression in one patient, (iii) increases in the frequency of peptide-specific CTLs in three patients and a decrease in one patient, and (iv) successful induction of peptide-specific CTLs from four patients. These findings suggest that the SYT-SSX junction peptide is safe to use as a vaccine and also has the property to evoke in vivo immunological responses. With respect to the clinical efficacy, none of the enrolled patients showed signs of tumor remission. However, in the case 5 patient, tumors showed some dormancy during the vaccination period, in comparison to the other five patients in whom tumors grew rapidly from the early phase of the regimen. The case 5 patient had received one cycle of systemic chemotherapy four months before enrollment, making the possibility of residual chemotherapeutic efficacy unlikely. The other five patients, except in case 1, had received systemic chemotherapy one to two months before perticipation. Notably, frequency of circulating peptide-specific CTLs decreased in the case 5 patient, while CTL frequencies remained unchanged or increased significantly in the other four patients examined. Decrease in circulating CTLs in the case 5 patient may have resulted from accumulation of CTLs at the tumor sites, although biopsy of the tumors was not performed. Vaccination trials of fusion gene-derived peptides have been reported with BCR-ABL in 12 patients with chronic myelogenous leukemia[ 16 ], EWS-FLI1 in 12 patients with Ewing's sarcoma[ 17 ], and PAX3-FKHR in four patients with alveolar rhabdomyosarcoma[ 17 ]. In addition, Matsuzaki et al.[ 18 ] reported a case of a synovial sarcoma patient who were treated with autologous dendritic cells pulsed with a mixture of SYT-SSX junction peptides (8–16 mer). In these studies, tumor remission was noted only in one patient with Ewing's sarcoma where IL-2 was concomitantly administered. These findings together with the results of our present trial have indicated the limited therapeutic efficacy of natural junction peptides. In this regard, we discovered improved in vitro immunogenicity of the SYT-SSX junction peptide by the substitution of an HLA-A24 anchor residue (position 9)[ 14 ]. Clinical study of this anchor-substituted peptide is currently underway. Besides modification of the peptide itself, concurrent use of adjuvants and cytokines, and adoptive T cell or/and dendritic transfer should further improve the therapeutic efficacy[ 8 , 19 ]. Also, it is important to determine the appropriate timing of vaccination and proper endpoints of clinical studies. To monitor the immunological responses, we used HLA/peptide tetramer and in vitro CTL induction. Other monitoring procedures such as ELISPOT assay should provide further information. Nevertheless, the use of an internal control tetramer with HIV peptide added strength in this present comparative analysis. As shown in Table 2 and Fig. 4 , reactivity of circulating T cells to HIV peptide tetramer remained under the background level throughout the vaccination period. Due to the rarity and highly malignant nature of synovial sarcoma, three out of six patients failed to complete the six-time vaccination regimen. Such difficulty in continuation of vaccination has also been found in a trial of patients with Ewing's sarcoma and alveolar rhabdomyosarcoma[ 17 ]. Another limitation in the current study is lack of analysis on immunological significance of SYT-SSX variants (SYT-SSX1 and SYT-SSX2). This question should be addressed in a larger scale analysis. Conclusions This is the first clinical trial of SYT-SSX fusion gene-derived peptide in patients with synovial sarcoma. The present trial demonstrated the safety and immunogenic property of the peptide. Modification of both the peptide itself and the related protocol is required to further improve the therapeutic efficacy. Abbreviations CTL; cytotoxic T lymphocyte, HLA; human leukocyte antigen, MHC; major histocompatibility complex, PBMC; peripheral blood mononuclear cell Competing interests The author(s) declare that they have no competing interests. Authors' contributions SK1 and YS carried out vaccinations and clinical evaluations. KI, TT, SK1,2, HA and KS carried out monitoring procedures. TW, SN, HS, HI, TT, HH, TI, ST, NS, and TY made substantial contributions to the design of the study.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545052.xml
517927	Functionally specified protein signatures distinctive for each of the different blue copper proteins	Background Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint. The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences. Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes. The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests. Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins. The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands [each from histidine] and one sulphur containing thiolate ligand [from cysteine], with strong interactions between the copper center and these ligands. Results Sequences that share such conserved motifs are crucial to the structure or function of the protein and this could provide a signature of family membership. The blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, dicyanin, umecyanin, uclacyanin, cusacyanin, rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, amicyanin and nitrite reductase which were identified in both eukaryotes and prokaryotes. ClustalW analysis of the protein sequences of each of the blue copper proteins was the basis for designing protein signatures or peptides. The protein signatures and peptides identified in this study were designed involving the active site region involving the amino acids bound to the copper atom. It was highly specific for each kind of blue copper protein and the false picks were minimized. The set of signatures designed specifically for the BCP's was entirely different from the existing broad spectrum signatures as mentioned in the background section. Conclusions These signatures can be very useful for the annotation of uncharacterized proteins and highly specific to retrieve blue copper protein sequences of interest from the non redundant databases containing a large deposition of protein sequences.	Background Most proteins can be grouped, on the basis of similarities in their amino acid sequences, into a limited number of protein families. Proteins or protein domains that belong to a particular family usually share functional attributes and are derived from a common ancestor. Highly conserved sequences in protein families are generally important for the function of a protein and/or for the maintenance of its 3-dimensional structure. Within the last decade, the sensitivity of sequence searching techniques has been improved by profile or motif-based analysis, which uses information derived from multiple sequence alignments to construct and search for sequence patterns [ 1 - 4 ]. By studying constant and variable properties of such groups, a signature for a protein family or domain can be derived which distinguishes its members from all other unrelated proteins. The problem of fast exact and approximate searching for a pattern that contains classes of characters and bounded size gaps (CBG) in a text has a wide range of applications, among which a very important one is protein pattern matching [ 5 ]. Unlike single-sequence similarity, a profile or motif can exploit additional information, such as the position and identity of residues that are conserved throughout the family, as well as variable insertion and deletion probabilities [ 6 ]. These signatures can be used to assign a newly sequenced protein to a specific family to formulate hypotheses about its function. By doing a keyword search, the protein sequences mined out from different databases is highly varied owing to different levels of redundancy. This could be due to the different strengths and weaknesses underlying the analysis algorithms used in different databases. The usage of the broad range signatures existing in databases, for the retrieval of blue copper proteins like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein brings out different kinds of copper proteins and a lot more of unrelated proteins. A search once again becomes necessary for sorting out the required blue copper proteins. The usage of pattern database would be more selective as it can identify family members based on the conserved functional region patterns. Keeping these broad spectrum signatures in mind, more specific and targeted protein signatures for each of the blue copper proteins was designed. The diagnostic success of these specified signatures over the wide range signatures mentioned lies in the number of true positives picked over the minimal or nil false positives picked from the non redundant databases. Blue copper proteins, which are also known as cupredoxins, are small, soluble proteins (10 – 14 kDa) whose active site contains a type 1-copper [ 7 ]. All these type 1 blue copper proteins possess an eight stranded Greek key beta barrel or beta sandwich fold and have a highly conserved active site architecture. The type 1 blue copper proteins exert their function by shuttling electrons from a protein acting as an electron donor, to another acting as an electron acceptor in various biological systems such as bacterial and plant photosynthesis [ 8 , 9 ]. During the electron transfer process, the copper ion changes from a diamagnetic Cu(I) to a paramagnetic Cu(II), oxidation state [ 10 ]. The coordination of the copper is determined by the conformation of its three closest ligands, two histidine nitrogens and a cysteine sulfur and of a fourth more distant ligand a methionine sulphur [ 11 ]. The coordination sphere of copper ions in blue copper protein rusticyanin is shown for example in Figure 1 . Type 1 copper sites are characterized by an intense blue color due to copper bound to thiolate [ 12 ]. An absorption is seen at 600 nm and gives rise to an unusual EPR signal, arising from asymmetrical copper site. Most of the cupredoxins have similar redox potentials ranging from 260 to 375 mV and function at pH values ranging from 6 to 8 [ 9 ]. Rusticyanin is an exception in having a very high redox potential of 680 mV [ 13 ]. The use of active site patterns or signatures is very rapidly becoming one of the essential tools of sequence analysis [ 14 , 15 ]. Although there is an appreciable amount of divergence in the sequences of the different blue copper proteins, the copper ligand sites are conserved. Direct application of the functionally specified signatures in databases, would help in quick retrieval of protein sequences related to that signature. The protein sequences thus retrieved were found to be highly specific to a particular blue copper protein. These signatures being highly specific allow the efficient mining out of uncharacterized proteins from the vast sequences deposited in different databases. Results Differentiation of blue copper proteins based on source of origin and active site tabulation The eukaryotic blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, umecyanin, uclacyanin, and cusacyanin. The prokaryotic blue copper proteins were rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, auracyanin, amicyanin and blue nitrite reductase. Plastocyanins are found both in eukaryotes and prokaryotes. Table 1 and 2 describe the active site functional region for each of the blue copper proteins mentioned above. The active site functional region indicates the aminoacids in the respective blue copper proteins bound to the copper atom. For example in plantacyanin with the protein data bank id 1F56, histidine at the 34 th position, cysteine at the 74 th position, histidine at 79 th position and methionine at the 74 th position are bound to the copper atom involved in electron transport chain. Keyword search for the specified blue copper proteins in different databases The number of sequences retrieved for a protein from different databases by keyword search are tabulated in Table 3 . As seen from Table 3 , a keyword search is no longer effective and precise in retrieving sequences of a particular kind. If still used, it is only a time consuming process, as the particular protein of interest has to be filtered from the retrieved sequences once again. For example, a varied response of data output is seen on a keyword search for plastocyanin. The sequences retrieved from each of the database in terms of number of protein sequences is 901 sequences in NCBI, 41 sequences in SwissProt, 10 sequences in TrEMBL, 350 sequences in Protein Information Resource, 375 sequences in EMBL, and 41 sequences in PDB. A search for the existing signatures for the blue copper proteins The signatures already available for each of the blue copper proteins retrieved from the Prosite motif database are listed in Table 4 . The number of protein sequences retrieved in response to the input of the already existing signatures for blue copper proteins in the PIR nREF database is shown in Table 5 . An overview of the results in Table 4 indicates that most of the blue copper proteins have a type 1 blue copper signature with an id PS00196. The multiple copper oxidase signature present in rusticyanin as shown in Table 4 with id PS00079 and PS00080 retrieves out 799 and 366 sequences respectively as shown in Table 5 . The existing rusticyanin sequences are very few in actual number and hence a secondary search becomes necessary. Even if the signature has annotated an unknown protein such as rusticyanin, it has to be searched amongst the 779 and 366 sequences retrieved. From Table 4 it is seen that plastocyanin has cyt b-heme (PS00192), cyt b QO(PS00193), photosystem1 PSAAB (PS00419), photosystem1 PSAGK(PS01026), Reiske 1 (PS00199) and Reiske 11(PS00200) as the signatures. As the names of the signatures suggest they are highly broad spectrum. The number of sequences picked out by these signatures as shown in Table 5 clearly indicates that most of these signatures are picking a lot more of other sequences other than plastocyanin and some of the signatures are missing out some plastocyanin sequences. Designing functional protein signatures for the different blue copper proteins As shown in 'Appendix 1 [see Additional file 1 ]' the newly designed signatures and peptides based on the ClustalW results for plastocyanin sequences of both eukaryotic and prokaryotic origin are shown in Table 6 . The same procedure as shown in Appendix 1 [see Additional file 1 ] was followed for the other blue copper proteins and the newly designed signatures and peptides based on the ClustalW results are shown in Table 6 . The new signatures and peptides thus designed for the different blue copper proteins, picked out highly specific sequences from the PIR nREF database consisting of sequences from the PIR [282,987 sequences], SwissProt [146,720 sequences], TrEMBL [1,069,649 sequences], GenPept [1,724,186 sequences], Ref Seq [756,736 sequences] and PDB [24,863 sequences]. The number of sequences retrieved based on the new signature for each of the blue copper protein is shown in Table 6 . Discussion The members of a protein family can be identified by collecting the matching sequences to profile or motif databases. Protein signatures are sequence motifs diagnostic to a protein family indicating function. Signatures are matched to protein sequences in the non redundant databases and is scored using a dynamic programming algorithm which permits permeability in gap distance and residue type [ 16 ]. Generating a signature involves identifying residues in a protein sequence that imparts functional properties to the protein. Protein signatures are efficient miners of related protein sequences having the same functional residues, which belong to the same class of proteins from the abundant sequences present in the non redundant databases. All the copper ions in the living cells are protein bound, as it is toxic in its free form. In most copper proteins, the copper ion having the ability to change valence state is mainly involved in catalysis of biological process, or the transport of electrons different proteins in a cell. Blue copper proteins also known as cupredoxins, have a type I copper site. They possess a single copper functional domain. The coordination of copper in most of the blue copper proteins is determined by the conformation of its three closest ligands, two histidine nitrogens and a cysteine sulfur and of a fourth more distant ligand a methionine sulfur [ 11 ]. In the case of auracyanin, stellacyanin and umecyanin the methionine is substituted by a glutamine residue, which binds as the fourth ligand to the copper atom. By doing a keyword search, we get varied results from the different databases as indicated in Table 3 owing to different levels of redundancy. On using a functionally related protein signature only relevant related sequences are picked out from the non redundant database as seen from Table 6 . Thus protein signatures can play a great role in extracting out highly related sequences from different databases than keyword searches. The signatures already available for the blue copper proteins like the Cyt b/b6, Photosystem 1 PSAGK, Rieske Iron Sulfur protein, and type I copper blue signatures are broad spectrum signatures. PS00196 a type I blue copper signature which is an already existing signature, when fed in the PIR nREF database has picked out 589 sequences as indicated in the result in Table 5 . We have also ensured that the active site region involving amino acids bound to the copper atom is present in all our signatures. Protein signatures designed taking into account the active site region will be very efficient for annotation of uncharacterized proteins. In one study the authors have used metal binding patterns of metalloproteins present in Protein Data Bank to search gene banks for new metalloproteins [ 17 ]. The protein signatures in a way can be compared to primers used for amplification. The more specific and concise a primer, the more specific is the amplification, similarly more specific the protein signature more significant are the picks from the non redundant databases. Specific signatures in a way reduce the time taken to pool related sequences from the abundantly available sequences from the non redundant databases. For example as shown in Table 4 the Type 1 blue copper signature is present in plantacyanin, stellacyanin, umecyanin, cusacyanin, halocyanin, azurin, auracyanin and nitrite reductase amongst the sixteen different blue copper proteins. When this signature is used as a query in the Prosite database even if an unknown protein is annotated it can only be as a type 1 blue copper protein, but it cannot classify it as a particular blue copper protein. The newly designed signatures or peptides will help in classifying the uncharacterized protein to the exact subtype of blue copper proteins. In this study, we have assigned functional property based signatures, which have the amino acid residues binding to the copper atom. It may be concluded that we have been successful in designing functionally related protein signatures for the blue copper proteins. Conclusions Signatures designed around the functionally important regions of a protein are valuable for annotation. In this study, specific signatures were designed around the active site regions of each of the blue copper proteins plantacyanin, plastocyanin, uclacyanin, stellacyanin, rusticyanin, sulfocyanin, amicyanin, halocyanin, pseudoazurin, azurin and nitrite reductase. These will be very useful for annotating uncharacterized proteins as blue copper proteins. Further, because of their high specificity to each subclass, they can be used in classifying the various subtypes of blue copper proteins. Methods Differentiation of blue copper proteins based on source of origin and active site tabulation The blue copper proteins were distinguished based on the source of origin as prokaryotic and eukaryotic. The active site residues of the eukaryotic and prokaryotic blue copper proteins, which bind the copper metal atom, were identified from the Protein Data Bank and tabulated. Keyword search for the specified blue copper proteins in different databases The name of each of the blue copper protein was given as a query in the keyword searches at NCBI, SwissProt, TrEMBL, PIR, EMBL and PDB databases to check for the number of sequences retrieved from each database and the results were tabulated. A search for the existing signatures for the blue copper proteins The signatures already existing for each of these blue copper proteins were identified from the Prosite motif database and tabulated. These already existing signatures were used as query patterns in the PIR Motif/Peptide match and a search was made against the PIR-nREF database. The PIR-nREF database consists of sequences from the PIR [282,987 sequences], SwissProt [146,720 sequences], TrEMBL [1,069,649 sequences], GenPept [1,724,186 sequences], Ref Seq [756,736 sequences] and PDB [24,863 sequences]. The number of protein sequences matching the query (already existing signatures for the blue copper proteins) was retrieved and the query results were tabulated. Designing functional protein signatures for the different blue copper proteins Each of the blue copper proteins from different sources were chosen from the different sequence databases and aligned using the multiple sequence alignment tool ClustalW. Conserved regions, which include the amino acids bound to the copper, reflecting the active site imparting a vital biological role of electron transport were chosen to design signatures. In regions showing 100% conserved sequences they were identified as conserved peptides. An example of how the functional protein signatures were designed is shown for plastocyanin in Appendix 1 [see Additional file 1 ]. The signatures and peptides were then submitted to the Protein Information Resource [PIR nREF] database for the protein pattern and peptide match and the results were tabulated. Authors' Contributions PG, AVG and SA participated in the design and coordination of the study. AVG carried out the bioinformatics search, the designing of the signatures and drafted the manuscript. PG and SA read and approved the final manuscript. Supplementary Material Additional File 1 Designing protein signatures: Illustrated example Plastocyanin. Plastocyanin sequences of eukaryotic and prokaryotic origin were retrieved from the PDB and SwissProt databases. The eukaryotic sequences were subjected to a ClustalW multiple sequence alignment. Signatures were designed based on the conserved pattern around the active site region [copper binding to four amino acids in plastocyanin]. The same procedure was adopted for plastocyanin sequences of prokaryotic origin. The newly designed signatures were used as queries in the Pattern/peptide match search at the PIR database [Protein Information Resource]. The numbers of plastocyanin sequences retrieved are tabulated in Table 6. The results were compared with the already existing signatures for plastocyanins and the number of sequences that these signatures picked up from the PIR database [data shown in Table 4 & 5]. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC517927.xml
544358	Modelling the impact of climate change on woody plant population dynamics in South African savanna	Background In Southern Africa savannas climate change has been proposed to alter rainfall, the most important environmental driver for woody plants. Woody plants are a major component of savanna vegetation determining rangeland condition and biodiversity. In this study we use a spatially explicit, stochastic computer model to assess the impact of climate change on the population dynamics of Grewia flava , a common, fleshy-fruited shrub species in the southern Kalahari. Understanding the population dynamics of Grewia flava is a crucial task, because it is widely involved in the shrub/bush encroachment process, a major concern for rangeland management due to its adverse effect on livestock carrying capacity and biodiversity. Results For our study we consider four climate change scenarios that have been proposed for the southern Kalahari for the coming decades: (1) an increase in annual precipitation by 30–40%, (2) a decrease by 5–15%, (3) an increase in variation of extreme rainfall years by 10–20%, (4) and increase in temporal auto-correlation, i.e. increasing length and variation of periodic rainfall oscillations related to El Niño/La Niña phenomena. We evaluate the slope z of the time-shrub density relationship to quantify the population trend. For each climate change scenario we then compared the departure of z from typical stable population dynamics under current climatic conditions. Based on the simulation experiments we observed a positive population trend for scenario (1) and a negative trend for scenario (2). In terms of the projected rates of precipitation change for scenario (3) and (4) population dynamics were found to be relatively stable. However, for a larger increase in inter-annual variation or in temporal auto-correlation of rainfall population trends were negative, because favorable rainfall years had a limited positive impact due to the limited shrub carrying capacity. Conclusions We conclude that a possible increase in precipitation will strongly facilitate shrub encroachment threatening savanna rangeland conditions and regional biodiversity. Furthermore, the negative effects found for positive auto-correlated rainfall support current ecological theory stating that periodically fluctuating environments can reduce population viability because species suffer disproportionately from poor environmental conditions.	Background In order to assess biodiversity response under climate change, Hannah et al. [ 1 ] recently emphasized the need to apply simulation models operating on a regional scale. Moreover, species respond differently to climate change because of different adaptations to their environment [ 2 ]. As a consequence, single-species models with a regional focus are essential to fully understand the manifold impact of global climate change. However, even though recent simulation tools have occasionally been applied for climate-sensitive animal species (e.g. [ 3 - 7 ]), spatial plant population models are extremely scarce (see review in [ 8 ]). In this study we show how climate change may affect the long-term population dynamics of the raisin bush, Grewia flava DC, a common, fleshy-fruited, woody plant species of South African savannas. Understanding the population dynamics of Grewia flava is a crucial task, because it is widely involved in the shrub/bush encroachment process (e.g. [ 9 , 10 ]). Shrub encroachment, i.e. the increase in woody plant cover, is a major concern for conservation and savanna rangeland management, due to its adverse effect on livestock carrying capacity (e.g. [ 11 - 13 ]) and biodiversity (e.g. [ 14 ]). In the context of global climate change, increase in woody plant cover has primarily been investigated in association with elevated CO 2 (e.g. [ 15 , 16 ]). For example, Bond et al. [ 16 ] suggested that higher rates of atmospheric CO 2 will have a positive effect on the post-fire regrowth of woody plants resulting in an increase in woody plant cover. Unfortunately, appropriate data verifying this hypothesis are not yet available. Here, we present a different approach. We used Spatial Grewia Model (SGM) (see [ 17 , 18 ]), a stochastic, spatially-explicit computer model to evaluate the impact of precipitation pattern change on Grewia flava population dynamics. In semi-arid and arid savannas, rain is the most important environmental parameter governing crucial life history processes in woody plants [ 19 ]. Hence, climate change related shifts in precipitation pattern will potentially have severe consequences for woody plant population dynamics. As a regional focus for our study we considered the southern Kalahari, the near centre of Grewia flava 's distribution. In this area, recent climatological studies proposed either a decrease in mean precipitation of 5–15% by the year 2050 [ 20 ], or an increase by up to 30–40% (e.g. [ 21 ]. Further studies suggest an increase in the frequency and variability of extreme rainfall events (e.g. [ 22 ]), as well as alternating phases with low and high rainfall, typical for Southern Africa [ 23 ]. The large divergence between the various precipitation scenarios raises the question how woody plants would react along this spectrum. Therefore, we have set up a systematic sensitivity study to explore Grewia flava population dynamics along precipitation gradients, so as to detect possible thresholds in the response of this species to continuous variations of a crucial environmental driver. Results By implementing relevant ecological processes of Grewia flava population dynamics into the SGM (see Figure 1 and model description in method section), we developed a standard scenario, based on annual rainfall for the period 1940 – 2000. This standard scenario led to stable population dynamics (see Figure 2 and method section). To compare population trends between four climate change scenarios we then used simple linear regression to calculate the z -value, i.e. the slope of the year – shrub density relationship. Figure 1 Flow chart of SGM showing causal pathways of Grewia flava population dynamics. Bold arrows indicate processes where population parameters and variables are affected by the annual type of rainfall (for detailed model description see text). Figure 2 Typical SGM time series of the standard scenario with 500 annual time steps. Population trend is given as shrub density with simple linear regression y = 0.0032x + 15.73. Mean slope of population trend for 100 simulation runs was z = 0.000049x + 13.41 with standard deviation of +/-0.017. Further model output from top to bottom: number of high (black) and low rainfall (grey) years per decade, fire occurrence (indicated as a black bar), density of fire- and drought-killed shrubs and density of recruits. Decades with frequent high rainfall years are indicated by frequent fires and increased fire mortality. Decades with exceptionally low rainfall show low fire frequency and increased drought mortality. Peaks of recruitment occur mostly in years with high rainfall and absence of fire. (1) Increase in precipitation An increase in the frequency of high rainfall years resulted in an increase of the z -value (Figure 3 and Figure 4 ). For example, a 20% increase in high rainfall years yielded a mean z -value of 0.01 (Figure 4 ). With an increase of 50%, the maximum z -value was 0.02. This upper limit could be clearly assigned, because the carrying capacity of cell type T had been reached, i.e. no further recruits were able to establish in the sub-canopy of trees. Even though additional juveniles may still emerge in cell type M , they are prevented from reaching the open due to a low probability of P matrix . Additionally, fire mortality is higher in cell type M . Figure 3 Typical time series for climate change scenarios with z -value of the population trend: increase in precipitation (1); decrease in precipitation (2); increase in inter-annual variation of precipitation (3); increase in temporal auto-correlation of precipitation (4) for period length PL with 20 and 250 years. Columns indicate 20%, 60% and 100% variation, respectively (see parameter values in Table 1). Figure 4 Results of simulation experiments for climate change scenarios according to: increase in precipitation (1), decrease in precipitation (2) and increase in inter-annual variation of precipitation (3) given as mean population trend z for 100 simulation replicates. The mean population trend is given as z for 100 simulation replicates plotted against percent variation in the probability of occurrence of extreme rainfall years as compared to the standard scenario. (2) Decrease in precipitation A decrease in mean precipitation resulted in a negative population trend (scenario 2, Figure 3 ) with a lower limit of z at -0.027 (Figure 4 ). A 10% increase of P low , corresponding to a probability of occurrence of low rainfall years of P low = 0.165, yielded a mean z value of -0.008. This continued to z = -0.02 for a 40% increase of P low . Above a 60% increase of P low 30% of all replicate runs resulted an extinction of the Grewia flava population within the 500 year time frame (not shown, however compare Figure 3 ). Comparing the upper and lower limits of the z- value for scenario (1) and (2), negative effects associated with an increase in drought events were more effective than the positive effects related to higher recruitment rates associated with comparable rainfall excess (0.02 as opposed to -0.027 see Figure 4 ). (3) Increase in inter-annual variation of precipitation An increase in inter-annual variation between low and high rainfall years below 100% did not cause any significant departure from stable population dynamics. However, above a threshold value of 100%, corresponding to a reduction of P average from 0.72 to less than 0.44 (see Table 1 ), we observed a decrease in the mean z -value. For example, for an increase in variation of 150%, z was -0.02 (Figure 4 ). Here, rainfall almost exclusively occurred as either high or low (P low = 0.375, P high = 0.325). Considering the negative and positive effects of low and high rainfall years, negative effects, such as drought, outweighed positive effects associated with enforced recruitment in high rainfall years. Table 1 Probability values for low (first value) and high rainfall years (second value) for climate change scenarios: increase in precipitation (1); decrease in precipitation (2); increase in inter-annual variation of precipitation (3); increase in temporal auto-correlation of precipitation (4) with 'good' phase (4 a ) and 'poor' phase (4 b ). Columns indicate 20%, 60% and 100% variation in the probability of occurrence of extreme rainfall years, respectively, as compared to the standard scenario (P low = 0.15, P average = 0.72, P high = 0.13). Scenarios / Variation 20 % 60 % 100 % (1) P low 0.150 0.150 0.150 P high 0.156 0.208 0.260 (2) P low 0.180 0.240 0.300 P high 0.130 0.130 0.130 (3) P low 0.180 0.240 0.300 P high 0.156 0.208 0.260 (4 a ) 'good' phase P low 0.120 0.060 0.000 P high 0.156 0.208 0.260 (4 b ) 'poor' phase P low 0.180 0.240 0.300 P high 0.104 0.052 0.000 (4) Increase in temporal auto-correlation of precipitation Despite constant mean and inter-annual variation in precipitation, increasing period length ( PL ) and intra-cycle variation ( ICV ) led to negative population trends and a decrease in z -value (Figure 5 ). Here, we found varying ICV thresholds for the PL scenarios tested, i.e. an abrupt departure from stable population dynamics. For example, a 10-year alternating rainfall cycle with an increase of 80 % in ICV (corresponding to a 5-year 'poor' phase with P low = 0.27 / P high = 0.026 and a 'good' phase with P low = 0.03 / P high = 0.234) yielded a z -value of -0.003, whereas population dynamics were stable for an ICV of 70%. Further thresholds were found above 60% ICV for a PL value of 20 years, 50% for 50 years, 40% for 100 years and 15% for 250 years. In the latter case, a large proportion of the population became extinct below 50% ICV where z was close to its lower limit. It is noteworthy, that we found no significant difference between similar PL scenarios that began with different phase conditions. Figure 5 Results of simulation experiments for increase in temporal auto-correlation of precipitation (climate change scenario 4) according to a PL value of 10, 20, 50, 100 and 250 years. The mean population trend is given as z for 100 simulation replicates plotted against percent variation in the probability of occurrence of extreme rainfall years as compared to the standard scenario. Discussion Based on our simulation results, we have demonstrated that climate change predictions of precipitation pattern for the southern Kalahari may significantly affect Grewia flava population dynamics. This enables us to estimate the possible consequences for land use management and biodiversity due to the keystone function of Grewia flava in the shrub/bush encroachment process. What are the implications of climate change related population trends for regional biodiversity and rangeland condition? (1) Increase in precipitation A climate change related increase in precipitation is commonly rejected [ 20 ]). However, based on a 28-year atmospheric circulation model, Mason et al. [ 21 ] proposed an increase in mean annual rainfall of 30–40% for the southern Kalahari. Yet, due to the model's insensitivity to topography, the authors also emphasize uncertainties in their predictions. This scenario would result in a strong increase in shrub density as indicated by a positive mean population trend for the SGM simulation period (Figure 4 ). Here, fire and drought mortality rates are too low to compensate substantial increase in juvenile recruitment associated with high rainfall years. On rangelands with domestic livestock this process may be strongly enhanced: in an earlier model version it was shown that cattle feeding on the foliage of Grewia flava may disperse seeds into the open matrix vegetation through dung deposition and thus facilitate shrub encroachment [ 18 ]. In this case we propose that an increase in precipitation will result in a catastrophic deterioration of current rangeland conditions within several decades. As a further consequence, biodiversity will most likely decrease due to the homogenization of woody plant community structure [ 24 ]. An increase in structural homogeneity due to shrub encroachment has been shown to adversely affect species diversity in both plants and animals (e.g. [ 14 , 25 ]). (2) Decrease in precipitation The majority of climatological studies propose a decrease in annual precipitation of 5–15% by the year 2050 [ 20 ]. For a 10% increase in the probability of occurrence of low rainfall years (at the cost of average years) we found a negative trend for Grewia flava population dynamics (Figure 3 ). High frequency of low rainfall years resulted in an increase of drought-related mortality and the reduction of shrub density. Increases in the probability of occurrence of low rainfall years of more than 40% lead occasionally to population extinction within the SGM time frame. However, we believe that under natural conditions population dynamics of Grewia flava may also stabilize at a lower level, as suggested by its distribution in arid parts of the southern Kalahari where droughts are more frequent (e.g. [ 26 ]). Even though precipitation decrease may mitigate the risk of local Grewia encroachment, a strong reduction in shrub density may lead to cascade effects in the food chain because the fruits are an important component in the diet of animals and humans in an environment where food resources are otherwise scarce [ 27 ]. (3) Increase in inter-annual variation of precipitation The inter-annual variation in precipitation, i.e. frequency and magnitude of extreme rainfall events, has been predicted to increase with ongoing climate change within the next decades (e.g. [ 22 , 28 ]). Rates may range between 10–20% for the southern Kalahari [ 21 ]. For this scenario SGM produced no significant departure form stable population dynamics (Figure 4 ). Thus, we propose that the predicted increase in inter-annual variability will have a low impact on the natural population dynamics and shrub density of Grewia flava . However, this may differ for rangelands with high cattle grazing: an earlier model version showed that increase in the probability of extreme rainfall years can increase shrub encroachment if additional seed dispersal by cattle into the matrix is considered [ 18 ]. An increase in variability of more than 100% revealed a significant threshold behavior with a negative Grewia flava population trend. Above this threshold frequent droughts kill the offspring and inhibit emergence. Ecological theory generally suggest that population viability decreases with increase in environmental variability or stochasticity [ 29 ], because favorable events have a limited positive impact due to the carrying capacity, whereas unfavorable events have a full negative impact [ 7 ]. However, to our knowledge, the general character of this relationship has not been studied in detail yet. Thus, the pattern described here provides new insights into the relationship between environmental variability and the corresponding population dynamics. (4) Increase in temporal auto-correlation of precipitation Tyson [ 23 ] showed evidence of periodic, non-random rainfall oscillations with a period of ca. 18–20 years in Southern Africa. This distinct pattern has recently been associated with El Niño/La Niña phenomena and may increase under climate change [ 30 ]. Since we did not simulate actual total rainfall we used values of ICV , i.e. intra-cycle variation and PL , i.e. period length to evaluate the impact of climate change related increase in positive auto-correlation of rainfall. In our study region, Kruger [ 30 ] found an oscillation with a PL value of 22 years between 1955 and 1991. The ICV values used in our simulation experiments refer to an increase in the probability of an extreme rainfall category and are thus difficult to compare with the cyclic variability of actual total rainfall. However, we compared the ICV values from our model with departures of annual rainfall from the inter-annual mean of a typical rainfall oscillation provided by Kruger [ 30 ]. Based on this, we infer that an ICV value above 60% resembles a realistic rainfall variation between favorable and unfavorable periods of a 22-year oscillation. Although we did not find a negative impact on Grewia flava population dynamics for this scenario, a slight increase in ICV combined with an increasing length of El Niño/La Niña phases might have a significant negative impact on the population viability of Grewia flava (see Figure 5 ). This will have profound consequences for other organisms which depend on, for example, fruit provision. As a further corollary, our results confirm current ecological theory that positive auto-correlation with constant average and inter-annual variation of a driving environmental parameter can reduce population viability and lead to extinction (e.g. [ 17 , 31 , 32 ]). In terms of the internal model processes of SGM , this means that negative effects in drought years (i.e. drought mortality and lack of reproduction) outweigh positive effects in wet years (i.e. increased reproduction). This is noteworthy, as mean annual rainfall and inter-annual variation was kept constant. Until now, this has been primarily shown in solely theoretical studies with under-compensating population dynamics (e.g. [ 33 ]) or individual-based animal models (e.g. [ 7 ]). To our knowledge this is the first empirical-based, plant population model that showed evidence of population persistence being negatively influenced by positive auto-correlation through a periodically fluctuating environmental driver. Parameter sensitivity and predictive power of SGM In order to assess the predictive power of SGM it is important to discuss the inference of actual precipitation amounts on probability values. Threshold values for extreme rainfall years are based on expert knowledge and may vary depending on the plant species considered. Moreover, percentage changes in extreme rainfall years, as applied in our model, are not necessarily equivalent to percentages changes in mean annual rainfall. For example, an increase in 'high' rainfall years by 10% represents an increase in mean annual rainfall of approximately 10%, depending on actual precipitation amounts in each 'high' rainfall year. Thus, it is important to derive principal population trends from the SGM model results rather than absolute predictions. Another issue regards the temporal dimension: long-term SGM simulation periods deviate from short-term predictions of climate change. However, population trends applied for only a few decades may yield biased results due to the cyclicity of Grewia population dynamics (see [ 17 ]). Finally, we stress that potential deviations in the annual probability of drought mortality may modify the output of the model. For example, a sensitivity analysis showed that a 100% increase in drought mortality for adult shrubs decreased the model output by 10% (see [ 18 ]). Conclusions In this study we have shown that climate change may have severe and sometimes unexpected implications at a regional species level. A woody plant like Grewia flava is relatively long-lived, fire and drought-resistant. Intuitively, this would suggest a low climate change impact on Grewia flava population dynamics. However, we found that despite Grewia flava 's capability to survive in a harsh environment, it may be strongly affected when rainfall decreases as predicted, or increases in periodical fluctuations. This may also include possible range shifts in regional distribution which have not been studied here. Based on the model and the climate change scenarios analysed, it would be inappropiate to forecasts changes in the geographical distribution of Grewia flava , because SGM parameters have been estimated and validated for populations in the Kimberley region of the southern Kalahari. Moreover, current geographical distribution suggests that buffer mechanisms may facilitate survival in more arid parts of the southern Kalahari (e.g. through higher variability in emergence as an adaption towards lower frequency of high rainfall years). Decrease in rainfall may reduce the severity of Grewia flava encroachment in the southern Kalahari. However, increase in rainfall will likely enhance this process. A shift from typical open grassland with solitary trees towards wide spread, homogeneous Grewia flava thickets implies negative cascade effects for other species, resulting in the loss of biodiversity [ 24 ]. As a further consequence, local rangelands are likely to be reduced in their carrying capacity of domestic livestock with negative effects for economic sustainability. Moreover, a shift in precipitation pattern may have further consequences, such as the alteration of fire regimes, grass biomass production or woody plant carbon uptake. Methods A detailed description of the simulation model SGM and previous results have been presented elsewhere (see [ 17 , 18 ]). SGM has been previously validated with empirical data and simulates population dynamics of Grewia flava under specific land use, fire and rain scenarios in southern Kalahari semiarid savannas. However, to facilitate a better understanding of the current results we will briefly describe the study species and relevant aspects of the simulation model. The study species In the open savannas of the southern Kalahari, Grewia flava typically grows beneath the dominant tree species Acacia erioloba [ 34 ], because bird-mediated seed dispersal predominately confines new establishments to woody plant microsites. However, occasionally large individuals may be found in the open grassland matrix at former tree sites, suggesting high longevity of Grewia flava . Under high cattle grazing a substantial proportion of seeds may be distributed into the open matrix vegetation, since cattle feed on the foliage and fleshy fruits of Grewia flava [ 18 ]. This may result in a substantial increase in Grewia cover, particularly around boreholes (e.g. [ 10 ]). Grewia flava has excellent resprouting capabilities after fire [ 35 ] and low drought mortality rates [ 36 ]. Size class distributions suggest a demographic bottleneck in early life stages due to low rates of emergence and high juvenile mortality [ 36 ]. General model structure The computer model SGM (see [ 17 , 18 ]) represents a grid-based approach iteratively simulating population dynamics of Grewia flava in annual time steps for a period of 500 years. Based on empirical demographic and spatial data from the Kimberley region of the southern Kalahari (see [ 36 ]), an initial population of 15 shrubs ha -1 was distributed on a 200 × 200 cell grid, with each cell representing 5 m × 5 m of savanna vegetation. The SGM grid is developed in two layers: a landscape and a population layer. In the first layer micro-site types of the savanna vegetation may change in the course of time. In the second layer SGM simulates population dynamics of Grewia flava . A cell type was classified as either tree ( T ) (i.e. occupied by Acacia erioloba ) or matrix type ( M ) (i.e. grassland vegetation). However, it switched from T to M status when a tree died after a mean life span of 200 years. The converse occurred where a new tree establishes. Initial tree distribution and spatial recruitment pattern was random with a constant density of 5 trees ha -1 for the entire simulation period. In each time step, SGM simulated important life history stages, environmental conditions and the key ecological processes of Grewia flava , the majority of which are governed by annual rainfall patterns (see Figure 1 ). For example in the model, rainfall directly determines the likelihood of a fire, drought and fire mortality, fruit crop size, emergence and annual shrub growth rate. Rainfall Annual rainfall in the southern Kalahari is highly variable, with precipitation ranging between 200 and 700 mm yr -1 . Rain falls almost exclusively during summer (November to April) with an inter-annual mean of 417 mm for the period 1940 – 2000 (Kimberley airport, South African Weather Service 2001, unpublished data). For this period we defined a threshold value of 150 mm below and above the long-term mean to classify years into 'low', 'average' and 'high' rainfall years (thresholds of 267 mm yr -1 and 567 mm yr -1 , respectively) (see [ 18 ]). Accordingly, frequency of extreme rainfall years resulted in an annual probability of P low = 0.15 for low and P high = 0.13 for high rainfall years (P average = 0.72). Similar classifications have been applied in other studies, e.g. in a spatial simulation model of Acacia raddiana in the Negev Desert [ 37 ]. Fruit production and seed dispersal Fruit production rates are based on mean crop size of Grewia size classes and may vary depending on the annual rainfall (see [ 18 ]). Seed dispersal is mostly zoochorous and spatially aggregated with a large proportion of seeds deposited in woody plant microsites. It is a crucial factor for the population dynamics and long-term viability of Grewia flava [ 36 ] and represented by two parameters: probability of seeds removed from a shrub and deposited in cell type M , P matrix , and cell type T , P acacia . P matrix varies randomly per year between 0.1–0.01% and represents occasional seed distribution through, e.g. small mammals. P acacia refers to bird-mediated seed dispersal with a range of 1–5% in high rainfall years, 2.5–7.5% in average and 5–10% in low rainfall years. Based on estimates from empirical data [ 36 ], the assumption of relatively constant seed dispersal rates may be reasonable as the proportion of seeds removed is most likely to be higher in years of lower fruit set. Fruit production typically varies more than bird abundances, and unless birds switch in exactly compensatory fashion to other fruits to the degree that Grewia flava becomes less abundant, fruit removal may be higher in years of low population size. Emergence Even though microsite types differ markedly in micro-environmental conditions, they do not differ in emergence rates of Grewia flava seeds [ 36 ] suggesting a similar emergence probability for cell type M and T . However, depending on annual rainfall conditions, the probability of emergence in the model may vary between 1–2% in average and 2–4% in high rainfall years (no emergence occurs in low rainfall years). As survival of Grewia flava seeds in the soil is very low, seeds that do not emerge are assumed to die. Fire and drought The occurrence and intensity of a fire depends on the amount of rainfall, since precipitation determines annual grass biomass production and thus fuel load [ 38 ]. In the SGM , fire was simulated probabilistically for the entire grid and may only occur in high and average rainfall years. Average frequency of 7.9 years in the model is supported by fire intervals reported from similar savannah types [ 39 ]. The impact of fire varies spatially and demographically: a seedling in the matrix vegetation has a 95% chance of being killed in a fire, compared with 4% for adult shrubs which largely resprout in the following year. For T cells, fire mortality probability is 0% for adults and 75% for seedlings. These model assumptions are realistic, since grass fuel accumulation and fire severity beneath trees is less than in tree inter-spaces (e.g. [ 40 , 41 ]). In the SGM , the probability of drought mortality varies between life stages and the annual rainfall conditions (see also model description in [ 18 ]). Annual drought mortality for adults is restricted to low rainfall years with a probability of 3% for both cell types. This assumption is based on data from O'Connor [ 42 ] and Schurr [ 36 ]. For seedlings the annual drought mortality probability is 90% for average and 50% for high rainfall years (see [ 18 ]). All seedlings are assumed to die in years with low rainfall conditions. Growth In the model, each shrub was assigned a size class with an average canopy cover. For definition of shrub size classes we used the canopy volume to group Grewia flava individuals into categories of small ( Grewia S ; <1 m 3 ), medium-sized ( Grewia M ; 1–10 m 3 ) and large plants ( Grewia L ; >10 m 3 ) as well as seedlings ( Grewia seedling ; temporary state after emergence, transforming to Grewia S with the following year). We assumed a maximum age for each size class member with MaxAge S = 5 years for small and MaxAge M = 25 years for medium-sized shrubs. These estimates are based on annual shoot growth rates and aerial photograph analysis of the study area (see [ 18 ]). In each annual time step an individual can accumulate a growth year and, if MaxAge is reached, attain the next size class with P transition ( S ) = 0.2 for small and P transition ( M ) = 0.1 for medium-sized shrubs. No growth occurs in low rainfall years. Carrying capacity and competition To incorporate intra-specific competition we used a simple causal approach that incorporates cell-based shrub cover. Therefore, we defined a carrying capacity of K = 25 m 2 as maximum total cell shrub cover. If shrub cover exceeded K , individuals in the cell died. Density-dependent mortality was simulated annually by removing the smallest individuals first, i.e. in descending order of size class, until shrub cover < K (for further details see [ 18 ]). Inter-specific competition between Grewia flava seedlings and the grass layer were neglected: empirical tests showed that emergence rates were similar within and outside of the grassy vegetation matrix (see [ 36 ]). For the adult stage, inter-specific competition with other woody plants is of low importance because Grewia flava often occurs as a mono-dominant species, particularly on bush encroached rangelands. Simulation experiments The standard scenario of the model, based on rainfall for the period 1940 – 2000, led to stable population dynamics (Figure 2 ). As standard deviation was generally high we performed 100 replicate simulation runs (± 0.0173 for the standard scenario, see Figure 2 ). To determine and compare population trends between the climate change scenarios we then used a simple linear regression of years vs. shrub density to calculate the z -value, i.e. the slope of the year – shrub density relationship. This is a reasonable approach for analyzing time-abundance relationships (e.g. [ 43 ]). When population dynamics were stable for 500 years, i.e. z was ≅ 0, the number of new recruits more or less resembled the number of fire- and drought-killed shrubs (Figure 2 ). Significant recruitment events mostly occurred in high rainfall years without fire. Based on the standard scenario we performed simulation experiments for each of the four different scenarios of climate change with variation in inter-annual mean of precipitation, coefficient of inter-annual variation, and temporal auto-correlation (see Table 1 ): (1) increase in precipitation, i.e. stepwise increase of P high resulting in a higher probability of high rainfall years (increase in recruitment events). Probability of low rainfall years is constant whereas frequency of average years is reduced, accordingly. (2) decrease in precipitation, i.e. stepwise increase of P low resulting in a higher probability of low rainfall years (increase in drought events), (3) increase in inter-annual variation of precipitation, i.e. stepwise increase of P low and P high resulting in a higher probability of low and high rainfall years at the cost of average years (unaltered inter-annual mean), (4) increase in temporal auto-correlation of rainfall with 'good' and 'poor' phases. For the scenario (4) we varied two parameters: (4a) introduction of rain cycles with increasing period length PL , i.e. longer phases of favorable and unfavorable rain conditions and, (4b) increase in intra-cycle variation ICV , i.e. increasing variation between alternating favorable and unfavorable periods within one rain cycle. For PL we applied a period length of 10, 20, 50, 100 and 250 years, respectively, with each period subdivided into a similarly long 'good' and 'poor' phase. For example, a 10-year period length was subdivided into a 5-year period of superior and a 5-year period of poor conditions. For an increase in ICV we alternately increased and decreased high and low rainfall probabilities in each period, respectively (see 4 a and 4 b in Table 1 ). For example, for an ICV value of 100% P low equaled 0.00 and P high 0.26 in a 'good' phase, whereas values were 0.30 and 0.00, respectively for a 'poor' phase. Through this procedure inter-annual mean and variation of rainfall probabilities were identical for each scenario and the default set of rainfall probabilities. Combined increase in PL and ICV resulted in an increase of positive auto-correlation. Near-decadal epochs of above- and below-normal rainfall have been identified for the period 1955–1991 [ 30 ] and may increase in ICV under predicted climate change. Author's contribution JT developed the SGM simulation model, conceived the study and drafted the manuscript. FJ supervised the study and the manuscript writing. Both authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544358.xml
535576	Getting a Whiff of Speciation by Reinforcement	null	Creating a new species is a bit like climbing a greased flagpole—it's hard to get started and even harder to keep going. Random genetic variations may introduce slight differences between two groups but, without some means to keep them apart, sexual interbreeding will quickly remix the genes and obliterate the differences. Accidents of geography—the rising of mountains or a course-changing river, for instance—can provide physical isolation, which then enables genetic divergence through the accumulation of mutations, either through natural selection or genetic drift. In contrast, speciation without geographic separation relies on the direct action of natural selection to complete the speciation process by strengthening behavioral differences, a process called reinforcement. One of the most powerful means of completing speciation is through the evolution of mate discrimination. A study by Daniel Ortiz-Barrientos and colleagues focuses on the genetic underpinnings of mate discrimination in Drosophila . They identify two loci that influence the likelihood that a female will choose to mate with a conspecific male, rather than one of a closely related species. Drosophila pseudoobscura and D. persimilis exist together along the west coast of the United States (sympatry), but separately elsewhere (allopatry). When together, they hybridize and produce sterile males. While D. pseudoobscura males will court females of both species, females prefer conspecific males. This female preference is stronger in sympatric females, an enhancement that presumably evolved by the direct action of natural selection to prevent females from wasting their reproductive efforts producing sterile sons. This variation allowed the authors to conduct a series of genetic crosses among flies of the same species but from different locations. Because the daughters of discriminating D. pseudoobscura females were just as discriminating as their mothers, Ortiz-Barrientos and colleagues concluded that female mating discrimination was inherited as a dominant trait. Further crosses showed that genes responsible for female preference were on the X and fourth chromosomes, and high-resolution mapping refined their locations sufficiently to allow the identification of likely candidate genes. While more work remains to be done, the most promising genes in both regions appear to be involved with olfaction. The fact that one of them, CG13982 , is known to up-regulate the other, bru-3 , strengthens the case that these are indeed promising candidates. Drosophila choosing their mates According to their findings, the authors propose a novel model of mating discrimination in D. pseudoobscura based on the combined response to auditory and olfactory cues. The first of these two layers, weak, or “basal” mating discrimination, has previously been associated with a set of traits for acoustic recognition and mapped to chromosomal regions that are inverted between the two species. Such inversions prevent recombination from purging alleles, thereby contributing to hybrid male sterility. As a consequence, when these species interbreed, they inexorably produce sterile males. The second layer, elucidated in the current study, is “reinforced” mating discrimination, which appears to be related to olfactory cues. This additional system of discrimination helps the first layer to fully eliminate the inevitable cost of producing sterile males. Once the nascent species have started up that slippery pole, reinforced discrimination could provide the traction needed to reach its top.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535576.xml
517714	Mutational dynamics of the SARS coronavirus in cell culture and human populations isolated in 2003	Background The SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome. The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations. Methods We sequenced complete SARS-CoV genomes taken from primary human tissues (SIN3408, SIN3725V, SIN3765V), cultured isolates (SIN848, SIN846, SIN842, SIN845, SIN847, SIN849, SIN850, SIN852, SIN3408L), and five consecutive Vero cell passages (SIN2774_P1, SIN2774_P2, SIN2774_P3, SIN2774_P4, SIN2774_P5) arising from SIN2774 isolate. These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates. Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated. Phylogenetic analysis was used to uncover molecular relationships between the isolates. Results Close examination of whole genome sequence of 54 SARS-CoV isolates identified before 14 th October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission. Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible. The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 × 10 -6 nucleotide substitutions per site per day (0.17 mutations per genome per day), or two mutations per human passage (adjusted R-square = 0.4014). Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are geographically associated: two Singapore isolates, one Taiwan isolate, and one North China isolate which appears most closely related to the putative SARS-CoV isolated from a palm civet. Non-synonymous mutations are centered in non-essential ORFs especially in structural and antigenic genes such as the S and M proteins, but these mutations did not distinguish the geographical groupings. However, no non-synonymous mutations were found in the 3CLpro and the polymerase genes. Conclusions Our results show that the SARS-CoV is well adapted to growth in culture and did not appear to undergo specific selection in human populations. We further assessed that the putative origin of the SARS epidemic was in late October 2002 which is consistent with a recent estimate using cases from China. The greater sequence divergence in the structural and antigenic proteins and consistent deletions in the 3' – most portion of the viral genome suggest that certain selection pressures are interacting with the functional nature of these validated and putative ORFs.	Background The Severe Acute Respiratory Syndrome (SARS) was first reported in November 2002 and rapidly spread to a number of distant global regions by early 2003. A new coronavirus, the SARS-CoV, was identified to be the cause of SARS [ 1 , 2 ] and was rapidly sequenced and characterized [ 3 , 4 ]. SARS-CoV is an enveloped, positive strand RNA virus with a wide host range. Recombination and mutation rates of RNA viruses are high, several orders of magnitude higher than DNA based microbes and in eukaryotes, and have been the cause of rapid changes in antigenicity, virulence, and drug sensitivity. Thus, the direct estimate of the mutation rates of the SARS-CoV in human populations and the analysis of the mutational spectrum would aid in developing strategies for monitoring and therapy. Previously, our analysis of 14 SARS sequences (five of which originated from Singapore) in May 2003 indicated that there are two different genotypes circulating in the world [ 5 ]. Recently, there has been a substantial increase in the number of SARS-CoV genomes sequenced. A total of 54 SARS-CoV genomic sequences (37 from the public database prior to October 14, 2003 and 17 sequenced within our institute) are used in our current analysis. This large dataset coupled with the availability of clinical data for cases related to Singapore patients and our molecular observations during in vitro cell passage presents an opportunity for a comprehensive analysis of the SARS-CoV mutational behavior. Methods Viral RNA genome isolation and sequencing SARS-CoV from the primary patient tissues were isolated by homogenizing the tissues in PBS buffer followed by a low speed centrifugation to obtain the viral particle containing supernatant. The virus-containing samples were also inoculated into Vero cell E6. The cells were maintained at 37°C using the usual viral cell culture media, and repassaged after 7 days of incubation. The virus-containing supernatants of homogenize or different passages of Vero cell E6 showing CPE were centrifuged at 23,000 RCF for 2.5 hours to pellet the viral particles and followed by RNA extraction using the QiAmp viral RNA mini kit (Qiagen, ). The RNA genome templates were converted into double strand cDNA and sequenced as previously described [ 5 ]. The processing of raw sequence reads (base calling, assembly, and editing) was done using PHRED/PHRAP/CONSED (University of Washington, Seattle, WA, USA, ). Genotype determination using MassArray technology A number of single nucleotide variations (SNVs) were further confirmed using a sensitive Mass Spectrometry based genotyping assay that was developed within our institute [ 6 ]. The RNA of the virus was first isolated using QiAmp viral RNA mini kit and then reverse-transcribed into cDNA (using the RNA as template, SuperScript kit from Invitrogen, and sequence specific primers), which were further purified. Primer extension assays were carried out for the SNVs of interest. The extension products were then detected in the MassARRAY (from Sequenom) to determine the genotypes. Data and statistical analysis We aligned the 54 SARS-CoV genomes using CLUSTALW [ 7 ]. To minimize the effect of sequencing errors and other artefacts to our analysis, we employed a filtering scheme where only SNVs shared by more than two different isolates are kept. The phylogenetic trees were reconstructed using the filtered variations. The reconstruction was done using PAUP* [ 8 ] with Maximum Likelihood criterion, keeping the other parameters to the default. The significance of the variations that pass the proposed mutation filter (where only mutations shared by more than 2 out of 54 isolates are considered real) can be assessed by calculating the probability that a random noise would meet the filtering criterion. The null hypothesis is that the noisy variations are generated independently between genomes. Let q be the rate of noisy mutation in a genome (based on our findings, as reported earlier in the text, we conservatively set , i.e. about two per SARS-CoV genome). The probability that, at a given nucleotide, a noisy mutation is shared by exactly i out of m different isolates is . Thus, the probability that a given nucleotide position has an erroneous mutation shared by more than k isolates is . In a genome with n bases, applying the Bonferroni inequalities, the probability that at least one position is corrupted by noise more than k times is p ( k , n , m ) ≤ n × s ( k , m ). In the case of 54 SARS-CoV genomes analyzed in this paper, , m = 54, k = 2, n = 30000, and hence the p -value of mutations that satisfy the filter is ≤ 2.2 × 10 -4 . In the estimation of SARS-CoV daily mutation rate, we employed the conservative constant mutation rate model [ 9 ], where the number of mutations d found in an isolate from its ancestor is proportional to the mutation rate k and the temporal difference t between the isolate and its ancestor, so that d = k × t . Based on the Singapore patients contact tracing information (see Figure 4 ), we obtained 6 pairs of isolates with known definite ancestor-descendant relationship, calculated the number of mutations ( d ) and the time difference ( t ) for each pair, and estimate the mutation rate k for the model using least square fitting. The goodness-of-fit were measured using the adjusted R-square statistics. Another pertinent question in the analysis of SARS-CoV evolution is prediction of the possible date of origin of the human SARS-CoV. Based on the animal-origin hypothesis of SARS-CoV, we assumed the SARS-CoV isolated from palm civet cat as the putative principal isolate that infected the human population. Adhering to the constant mutation rate model, we fit the following model: d x = d 0 + kx , where k is the daily rate of mutation, x is the sampling date measured relative to 1 st November 2002, and d x is the number of mutations, as compared to the civet cat isolate, of the isolate sampled at date x . Twelve data points were calculated and used to fit the model. The date of origin can be solved by solving x for d x = 0. The goodness-of-fit was measured using the adjusted R-square statistic. Results and discussion SARS-CoV mutations in vitro First, we sought to determine the rate of mutation of the SARS-CoV in Vero cell culture. To achieve this, we subjected SIN2774 isolate to 5 passages in Vero cells. At the appearance of cytolysis at each passage samples were withdrawn and their genomes completely sequenced. Any ambiguities by capillary sequencing were clarified by mass spectroscopic validation [ 6 ]. Our results showed that the Vero cell passages are actually comprised of two coexisting quasispecies bearing either an A or G at position 18372. No mutations emerged upon passage, and the ratio of A/G at 18372 remained constant over the passages (Table 1 ). This suggests that the mutation rate in culture of SARS-CoV is very low at <1 in 5 passages. Thus artificial mutations from limited in vitro cell culture are negligible. Mutations associated with human-to-Vero and Vero-to-human transition Next, we asked whether the transition from human tissue to growth in Vero cell culture engendered either mutations or clonal selection. The SARS-CoV were sequenced from three human tissue-Vero cell culture pairs of viral samples from Singapore and one pair was obtained from the public domain (see supplemental information, Table S1). The results shown in Table 2 showed that mutations emerged in only one case of human to Vero cell passage posted in Genbank (AS → HSR1) but in none of the Singapore pairs. However, the viral sequence from SIN3725V isolated from a lung sample showed evidence for co-infection by two distinct genotypes of SARS CoV. This was manifested by the simultaneous presence of T and C at positions 548, 1727, 13347, confirmed by genotyping using MALDI-TOF MassARRAY technology. Subsequent deconvolution by tracing the "haplotypes" at these loci in different Singaporean isolates revealed probable sequence signatures of T, T, C at these locations in one isolate and C, C, T in the other (see Table S2). Though tissue-derived SIN3725V has two SARS-CoV quasispecies, the isolate after subsequent Vero cell culture showed only one (bearing the T, T, C haplotype, supplemental information, Table S2). These results again show that coinfection by multiple quasispecies is not uncommon in human tissues, and that passage to Vero cells may either generate new mutations at a low rate, or titrates out one quasispecies in the transition. Singapore encountered an unusual incident where a stable lab SARS-CoV isolate commonly used for in vitro experimentation accidentally infected a laboratory worker [ 10 ]. We sequenced both the originating laboratory isolate (SIN_WNV; see Table S1) and the viral sample directly from the patient's sputum (SIN0409; see Table S2) and found no sequence difference between the two viruses. This reconfirms that the mutation rate from a single point source of virus has a low mutation rate when expanded during human infection. Sequence variation filter Inferring phylogenetic relationships between the known SARS-CoV isolates using existing public data has been problematic because of the potential for sequencing errors. Moreover, the rate of SARS-CoV mutation in culture was not previously known and was thought to be significant given the mutation rates in other RNA viruses. Our experiments provided information as to the potential causes and rates of sequence variations of the SARS-CoV in culture. Based on our Vero cells passages and human-to-Vero transition data, we estimated that, at most, one sequence variation from the original tissue virus can be accounted for by in vitro culture artifacts. The average base-calling error probability (as reported by PHREP [ 11 ]) of our sequences is about 7.5 × 10 -5 , or 2.25 errors per SARS-CoV genome. Accordingly, we suspected that sequencing errors in the reported SARS sequences would be approximately 1–2 bases per reported genome. We used this information to assess the true sequence variants reported in the public SARS sequence databases employing a "mutation filter" [ 5 ]. This mutation filter identifies a sequence variant as a probable mutation if it appeared in more than one isolate. Higher filter stringency can be applied by demanding a sequence variant to be present in two, three, or more isolates. A total of 54 isolates were analyzed, including 22 from Singapore. Our results show that the number of mutations appearing in only one isolate is high at 349 (see supplementary Table S3); however, those mutations present in more than two isolates are much lower and appeared relatively stable (Figure 1 ). Statistical analysis confirmed that the probability of finding any false mutation shared by more than two isolates out of 54 is very low ( p ≤ 2.2e - 4) as compared to the probability of finding a false mutation shared by more than one isolate ( p ≤ 0.19). These results are consistent with our error estimates as outlined above. We tested the biological validity of this approach by examining the mutational frequency of known genes in the SARS genome. Because of the importance of the 3CL protease and the polymerase for viral replication, we suspected that true non-synonymous mutations in the SARS-CoV present in clinical samples might be rare in these two ORFs in comparison to other structural genes such as those encoding the S, M, and N proteins. Without a mutation filter, sequence variations are commonly observed in the 3CL protease and the polymerase genes. However, when mutations are identified only as variants seen in two or more isolates, then no mutations are detected in the critical 3CL protease and polymerase genes, whereas mutations are noted in the S, M, and N genes regardless of the filter stringency (Figure 2 ). Therefore, we determined that the most effective mutation filter is presence of a sequence variant in more than two isolates. Molecular history of the viral isolates Using this filter stringency, we assessed the phylogenetic relationship between all 54 isolates describing the recent SARS epidemic. TOR2, Urbani, SIN2500, HKU-39849, CUHK-Su10 formed the core of the early isolates. Employing these as "root", four major clusters appeared: two Singaporean branches, one Taiwan branch, and one North China branch (Figure 3 ). Validating these clusters, the two sequences from Germany, 'Frankfurt' and FRA, which grouped with the Singaporean branch, were actually derived from the Singaporean doctor who treated the patient SIN2774 and was later hospitalized in Frankfurt. Of the Singaporean cases, SIN2500, SIN2677, SIN2748, and SIN2774 formed one molecular sub-branch which matched with the contact tracing (Figure 4 ). The clinical contact tracing data was ambiguous as to the direct source of SIN2679's exposure. Intriguingly, however, SIN2679 was the root of a second sub-branch within Singapore that had its origins most probably from the Hotel M cluster. This suggested a potential direct infection of SIN2679 from a Hotel M source other than SIN2500. Using the same contact tracing information, we calculated the average mutation rate during human transmission to be about two mutations per human transmission. Estimation of the mutation rate of the SARS-CoV We obtained the precise dates of symptom onset of 13 Singaporean cases (Table S1). Using the common mutations identified through application of the mutation filter, we employed the constant mutation rate model and estimated the mutation rate of the SARS-CoV during this recent epidemic. We estimated the mutation rate to be 0.1722 nucleotides per day, or 5.7 × 10 -6 nucleotide substitutions per site per day (adjusted R-square value of the fitted model = 0.4014). The rates for synonymous and non-synonymous mutations were equivalent at 2.5 × 10 -6 and 3.2 × 10 -6 nucleotide substitutions per site per day respectively. Using the Singapore isolates with known date of onset, and using the SZ3 and SZ16 genomes isolated from palm civet cat [ 12 ] as the putative "original" SARS-CoV that jumped from animal to human, we calculated the daily substitution rate to be 0.1303 nucleotides per day, or 4.3 × 10 -6 nucleotide substitutions per site per day, (adjusted R-square = 0.5880) and the estimated possible "date" of SZ3/SZ16 emergence was Oct 21, 2002. Overall, the mutation rate of SARS-CoV appears to be consistent with the reported rate of other viruses [ 13 , 14 ]. The mutational analysis also revealed 5 separate deletions and one insertion that distinguished the different isolates (Figure 5 ). Intriguingly, they all clustered within a short 200 bp region in the 3' end of the viral genome spanning putative ORFs sars 7b to sars 8b. Despite the overlapping nature of some of the deletions, there was no descendent relationship amongst them and the addition of the insertion/deletion information did not add to the clustering. Our assumption therefore is that this is a region of relative instability that is dispensable for viral replication. Conclusions The focus of this investigation was to measure the mutational frequency and dynamics both in vitro and in vivo of the SARS-CoV. Our findings suggest that the overall SARS-CoV's rate of mutation in culture is low. Inoculation of Human SARS-CoV into Vero cell introduces, on the average, less than one nucleotide mutation. Subsequent culturing of SARS-CoV infected Vero cells induced less than one nucleotide mutation in the five consecutive Vero cell passages. No mutations were also observed during the infection of SARS-CoV cultured in Vero cell to human. This would be consistent with the notion that the SARS-CoV isolates from the patients that gave rise to the in vitro lines are well adapted for in vitro growth. Our proposed mutation filter, which is based on these observations, seems to be stable and effective. Using this filter, reconstruction of molecular phylogenetic relations of the 54 SARS-CoV genomes revealed at least three major branches composed of cases related to Hotel M (Hong Kong), cases reported in North China, and cases found in Taiwan. Moreover, we show that these molecular sequence associations can be effectively used to more precisely reconstruct contact tracing. Our estimated the daily substitution rate of SARS-CoV to be 0.1722 nucleotides, or 5.7 × 10 -6 nucleotide substitutions per site, a mutation rate similar to other RNA viruses [ 13 , 14 ]. Taking the SARS-CoV isolated from palm civet cat as the putative originating SARS-CoV, our calculations suggest that the earliest possible date for SARS emergence is predicted to be Oct 21, 2002. During the final preparation of this manuscript two reports were published addressing the mutation rate of the SARS-CoV in human populations. Yeh et. al. [ 15 ], examining Taiwanese SARS samples estimated the CoV mutation rate to be about 1.83 × 10 -6 nucleotides per site per day. The Chinese SARS Molecular Epidemiology Consortium [ 16 ], examining a larger number of viral isolates, recently determined the mutation rate to be 8.26 × 10 -6 nucleotides per site per day using samples from China. These estimates were very close to ours. In addition, The Chinese Consortium [ 16 ] projected the time of emergence of the SARS CoV epidemic to be November 2002. The remarkable consensus of these three studies using different patient populations on the mutational dynamics of the SARS CoV suggests that these results are bona fide. Competing interests None declared. Authors' contributions VBV and ETL performed most of the data analysis and prepared the draft manuscript. STSY and LAE provided SARS-CoV samples from patients and Vero cells. KFT and OEE provided continue SARS-CoV containing Vero cell passage samples. YR and LWS designed the experiments and coordinated sample acquiring and the viral genome sequencing. CLW and TZ generated all DNA sequence data. WHL processed all DNA sequence and assembled the viral genomes. JL verified all sequence variations by conducting MALDI-TOF mass spectrometry analysis. PK assessed the effects of nucleotide variations to ORFs and their proteins. VBV and PML carried out the mathematical and statistical analysis. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Table S1 List of the sequences used in the analysis. This list is available at http://giscompute.gis.a-star.edu.sg/sars_mut_dyn/ Click here for file Additional File 2 Table S2 Single nucleotide variations (SNVs) detected initially in capillary sequencing (second row) and subsequently confirmed by MALDI-TOF MS-based genotyping (first row). This table is available at http://giscompute.gis.a-star.edu.sg/sars_mut_dyn/ Click here for file Additional File 3 Table S3 Complete list of single nucleotide variations (SNVs) observed in the 54 SARS-CoV isolates. The Singapore sequences used were all based on capillary sequencing. This data is available at http://giscompute.gis.a-star.edu.sg/sars_mut_dyn/ Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC517714.xml
434155	Host Immunity Escalates the Evolution of Parasite Virulence	null	Strictly defined, evolution is a change in the gene pool, or total set of genes, of a given population over time. Genetic changes that increase the fitness of an organism—that is, increase survival or fertility—are more likely to be retained, through natural selection, and passed on to succeeding generations. In the classic case of Darwin's finches, different ecological niches exerted different selective pressures on an original population, and resulted in 14 different species, each sporting a beak uniquely adapted to harvesting particular available food sources. When it comes to microbial evolution, an ecological niche often takes the form of a host. If the microbe is a pathogen, its presence might trigger strong selective pressure from the host's immune system, precipitating an evolutionary two-step between microbe and host. Hosts with strong immune defenses can typically tolerate relatively virulent pests: conversely, ill-defended hosts die, which is bad news for the parasite. When the myxoma virus first infected a population of European rabbits in Australia in 1950, the virus was particularly lethal. Over time, less virulent strains were selected for—killing off your habitat would be an unsustainable fitness cost by most standards—and the rabbits developed resistance. In keeping with evolutionary theory, host immunity should affect the evolution of parasite virulence. Though theory predicts that immunity could potentially heighten virulence, there's no evidence that this is true. Being able to predict how natural selection will act on, and thus shape, virulence is vital for developing effective public health policies—and desperately needed vaccines—to deal with the ever growing roster of rapidly evolving pathogenic threats. Malaria infecting red blood cells To investigate whether immune system defenses escalate pathogen virulence, Margaret Mackinnon and Andrew Read studied the malarial parasite Plasmodium in the mouse. Mackinnon and Read first directly injected two groups of mice with infectious parasites: “immunized” mice, which had been exposed to Plasmodium and then treated with the antimalarial drug mefloquine, and “naïve” mice, which had not. Parasites were serially transferred twenty times via a syringe from one mouse host to another. The virulence and infectiousness of the respective strains were evaluated by introducing the strains into another set of immunized and naïve mice. As theoretically predicted, parasites evolved in the immunized mice were indeed more virulent than parasites evolved in the naïve mice. But what if the parasites were first transmitted through their natural vector, the mosquito, rather than through a syringe? Would they be as virulent? Interestingly, infection was not as severe after mosquito transmission. But parasites evolved in the immunized mice retained a higher level of virulence than those evolved in the naïve mice. This means that immunity accelerates the evolution of virulence in malaria, even after mosquito transmission, making them more dangerous to nonimmunized hosts. How does immune selection create more virulent pathogens? One possibility is that even though many parasites die in immunized hosts, those that “win the race to the syringe”—or the mosquito—are likely genetically equipped to stay ahead of advancing immune system defenses. It's not entirely clear why selection would favor more virulent parasites, but since the virulent strains showed no problems transmitting infection to new hosts, it's likely that such strains would spread throughout an immunized population. While mosquito transmission likely plays a significant role in virulence evolution—it clearly reduced virulence here—the molecular mechanics of this effect are also mostly speculative at this point. Many questions remain, but these results make a strong case that vaccine development aimed at protecting individuals against infectious pathogens would do well to consider the evolutionary implications, or increased pathogen virulence could be an unintended consequence.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC434155.xml
555533	Delta activity independent of its activity as a ligand of Notch	Background Delta, Notch, and Scabrous often function together to make different cell types and refine tissue patterns during Drosophila development. Delta is known as the ligand that triggers Notch receptor activity. Scabrous is known to bind Notch and promote Notch activity in response to Delta. It is not known if Scabrous binds Delta or Delta has activity other than its activity as a ligand of Notch. It is very difficult to clearly determine this binding or activity in vivo as all Notch, Delta, and Scabrous activities are required simultaneously or successively in an inter-dependent manner. Results Using Drosophila cultured cells we show that the full length Delta promotes accumulation of Daughterless protein, fringe RNA, and pangolin RNA in the absence of Scabrous or Notch. Scabrous binds Delta and suppresses this activity even though it increases the level of the Delta intracellular domain. We also show that Scabrous can promote Notch receptor activity, in the absence of Delta. Conclusion Delta has activity that is independent of its activity as a ligand of Notch. Scabrous suppresses this Delta activity. Scabrous also promotes Notch activity that is dependent on Delta's ligand activity. Thus, Notch, Delta, and Scabrous might function in complex combinatorial or mutually exclusive interactions during development. The data reported here will be of significant help in understanding these interactions in vivo .	Background Notch (N) and Delta (Dl) are cell surface proteins that are required for differentiation of almost all tissues in the fruit fly Drosophila melanogaster . They are evolutionarily conserved, functioning similarly in animals from worms to humans [ 1 , 2 ]. The best-known instance of their function is the process of lateral inhibition that initiates differentiation of the neuronal and epidermal tissues from proneural cells that are predisposed to making the neuronal tissue. Proneural cells express high levels of the neuronal transcription co-factors from the Achaete Scute Complex (ASC) or related genes [ 3 , 4 ]. These factors require their partner Daughterless (Da) to activate transcription of the neurogenesis genes [ 5 - 7 ]. Da is expressed at low levels in all Drosophila cells [ 8 ] and up regulated in proneural cells specified to differentiate the neurons [ 5 ]. Whether or not the up regulation of Da expression is part of lateral inhibition is not clear in Drosophila. In Caenorhabditis elegans , however, the differential accumulation of the Da homolog HLH-2 is the earliest detectable difference between the cells taking up alternate fates during lateral inhibition [ 9 ]. As N and Dl are known to regulate Da expression [ 10 ], it is very possible that Da expression is regulated during lateral inhibition in flies as well. When N expressed on one proneural cell binds Dl expressed on the neighboring proneural cell, N is proteolytically cleaved to release the Notch intracellular domain (N intra ) from the plasma membrane. N intra translocates to the nucleus and, in association with the transcription factor Suppressor of Hairless (SuH), activates transcription of the Enhancer of split Complex (E(spl)C) genes. Cells that express a high level of E(spl)C RNA suppress their neuronal predisposition, become the epidermal precursor cells (EPCs), and differentiate the epidermis. Cells that express a low level of E(spl)C RNA and a high level of Da protein become the Neuronal Precursor Cells (NPCs) and differentiate the nervous system [ 1 , 2 , 5 , 11 ]. From here onwards, we refer to this SuH dependent N activity that promotes expression of E(spl)C RNA as SuH/N intra signaling. A 1.5 to 2-fold difference in the level of SuH/N intra signaling is sufficient to initiate specification of the EPCs and the NPCs [ 11 ]. This difference is amplified by subsequent activities of N and Dl, or activities of other genes responding to the initial difference in the level of SuH/N intra signaling. The lateral inhibition process described above is repeatedly used during development for differentiation of various tissues with minor variations or changes in target genes. Scabrous (Sca) is a secreted factor that is produced at high levels in the NPCs and functions non-autonomously to promote specification of the EPCs during differentiation of the compound eye and the bristle organ [ 12 , 13 ]. In its absence, lateral inhibition is not abolished but is reduced in strength or becomes imprecise indicating that Sca only refines the process. Sca binds N and stabilizes it. These actions promote formation of sharp boundaries between neuronal and non-neuronal cells during development of the compound eye [ 14 ]. The possibility that Sca might bind Dl as well is suggested by the observation that simultaneous over expression of Sca almost completely blocks the effect of Dl over-expression on wing margin development but hardly modifies the effect of N over-expression [ 15 ]. Dl and Sca have also been observed to co-localize in intracellular vesicles in vivo [ 13 ]. The observations that Sca can promote N activity [ 14 ] but block Dl activity are paradoxical as SuH/N intra signaling is very much dependent on the activities of both N and Dl. One explanation for this paradox could be, that Sca promotes lateral inhibition by having one effect through N and a different one through Dl. Therefore, we addressed the following questions in this study. Does Sca bind Dl? If yes, does it affect any Dl activity? Are there Dl activities independent of its activity as a ligand of N? Is Sca capable of activating N in the absence of Dl? N and Dl are expressed in almost all cells in vivo and N receptor activities in response to Dl binding are widely used during development. In developmental instances where Sca is present, the expression data suggest that both N and Dl will have access to Sca. Thus, it is very difficult to separate in vivo the activities of N alone, Dl alone, N on Dl, Dl on N, Sca on N, Sca on Dl, and Sca on N and Dl together. Therefore, we addressed the questions posed above in an in vitro model system based on Drosophila Schneider (S2) cells. S2 cells do not express the endogenous N, Dl, or Sca [ 14 , 16 ]. S2 cells expressing N (S2-N cells) mixed with S2 cells expressing Dl (S2-Dl cells) reproduce all aspects of lateral inhibition [ 16 - 22 ]. Using these cells and the medium prepared from S2 cells expressing Sca into the medium [ 14 ], we show that Sca binds Dl, Dl has activity independent of its activity as a ligand of N, Sca can affect this activity of Dl, and Sca can activate N in the absence of Dl. These observations would be useful for undertaking the challenging task of determining how the various activities of N, Dl, and Sca are integrated during tissue differentiation. Results Sca associates with Dl Although N and Sca complexes could be immuno-precipitated [ 14 ], we, and others [ 23 ], had failed to detect Sca on S2-N cells. We suspected that some factor present in the tissue culture medium was washed away when the cells were processed for immuno-fluorescent detection of Sca. To overcome such problems, we made Sca-GFP and established stable S2 cells expressing it (S2-Sca-GFP cells). S2-Sca-GFP cells produced the Sca-GFP protein of the expected size (as determined by western blotting) and both Sca and GFP antibodies recognized this protein (data not shown). We concluded that S2-Sca-GFP cells expressed the expected Sca-GFP protein and used the conditioned medium from these cells to treat live S2-N, S2-Dl, and S2 cells. Live S2-Dl cells showed the strongest GFP signals, followed by live S2-N cells, and then live S2 cells (Fig. 1A–C ). The signals were so strong on the S2-Dl cells that the signals on S2-N cells were not obvious at the same brightness/contrast settings. When cells were simultaneously fixed and rinsed with 1X PBS, the signals were comparable at the same settings (insets in Fig. 1A–C ). Signals could not be detected on S2-N or S2-Dl cells after three 5-minute washes with 1X PBS, confirming our suspicion that the standard immuno-fluorescence procedure is inappropriate for detecting Sca binding on S2-N or S2-Dl cells. Secreted GFP did not bind the surfaces of any of these cells (data not shown). This indicated that the Sca part of Sca-GFP fusion protein bound the S2-N and S2-Dl cell surfaces. In all experiments conducted to determine the activity of Sca, N, or Dl, that are described below, we used only S2 cells expressing the wild type Sca because (1) we do not perform washes to remove non-specifically bound proteins and (2) we wanted to avoid possible GFP associated effects (stability, etc.). Figure 1 Sca associates with Dl. A-C . Fluorescent photomicrographs of different cell lines treated with Sca-GFP medium for 30 minutes. Cells simultaneously fixed and rinsed in 4% paraformaldehyde/1X PBS are shown in the insets. Experiments were repeated three times. D . Western blots showing recovery of Dl in Sca immuno-precipitates from total protein extracts prepared from S2-Dl cells treated with S2-Sca cells. S2-Sca cells were used instead of Sca conditioned medium to maximize the ratio of bound to unbound Sca. Cross-linker = membrane insoluble and cleavable 3,3'- Dithiobis (sulfosuccinimidylpropionate) (DTSSP), which cross-links proteins interacting at the cell surface. IP Ab = antibody used for immunoprecipitation; W Ab = antibody used on the western blot; ppt = immunoprecipitate; super = supernatant. Experiments were repeated two times. We have previously shown that Sca and N form complexes [ 14 ]. To determine whether Sca forms complexes with Dl, we performed immuno-precipitation experiments with S2-Dl cells that were co-cultured with S2-Sca cells. Proteins interacting at the cell surfaces were either cross-linked or un-linked prior to cell lysis for protein extraction. Membrane insoluble cross-linkers improve recovery of cell surface complexes [ 18 , 24 ]. Sca immuno-precipitation recovered Dl strongly in the presence of cross-linkers and relatively weakly in the absence of cross-linkers (Fig. 1D ). No bands were observed when S2 cells were used instead of S2-Dl cells (data not shown). In the reverse experiments, Dl immuno-precipitations failed to recover Sca, possibly because there was too much unbound Dl in the extracts. Dl and Sca were not detected in the absence of immuno-precipitation antibodies (Fig. 1D , lanes 1 and 4) or in the absence of Scabrous (data not shown). We also recovered Sca in Dl immuno-precipitations and Dl in Sca immuno-precipitations from protein extracts of wildtype embryos (data not shown). These observations indicated that Sca associates with Dl. We explored the consequence of this association. Da expression in Dl cells is reduced in response to Sca N promotes expression of E(spl)C m3 gene in response to Dl [ 19 , 20 ]. We examined whether Sca promoted expression of E(spl)C m3 in S-N cells or S2-Dl cells and found that it was indeed the case with S2-N cells, but not with S2-Dl cells (Fig. 2A , lanes 1–6). S2-N cells showed a low level of E(spl)C m3 expression when S2-Dl or S2-DlΔI cells were replaced with S2 cells, in the absence of Sca (Fig. 2A , lanes 1, 7–8); S2-Dl or S2-DlΔI cells mixed with S2 cells did not show any accumulation (Fig. 2A , lanes 13–16). The low level of E(spl)C m3 RNA expression in S2-N cells in the absence of ligands is due to the low level of N intra produced upon induction of N expression in S2 cells [ 18 ]. This expression increases upon ligand treatment [ 18 ], resulting in increased expression of E(spl)C m3 RNA expression (Fig. 2A , lanes 2, 10, 12). Numerous repetitions of the experiments indicated that Dl is a more potent ligand of N than Sca with respect to induction of E(spl)C m3 expression (data not shown). Figure 2 Dl down-regulates Daughterless protein expression, and N up-regulates E(spl)C m3 gene expression, in response to Scabrous. A . Northern blots of total RNA from the indicated cell mixtures extracted at 0 or 45 minutes after treatment with medium containing Sca (+) or not (-). Gene probes used are shown on the right. m3 = E(spl)C m3 and rp 49 = a ribosomal protein gene used to show the levels of total RNA in the lanes in all northern blots. Sca = conditioned medium prepared from the S2-Sca stable cell line in all experiments here onwards. The control medium used along side Sca medium (-) was prepared from heat shocked S2 cells. Experiments were repeated two times. For unknown reasons, the medium collected from heat shocked S2 cells (used in lanes 1, 3, and 5) produced higher background levels of E(spl)C m3 RNA in S2-N cells (lane 1). B . Western blots showing the levels of Da and Dl in different Dl cell lines. S2-Dl, S2-Dl(1) and S2-Dl(2) are independently established hsDl cell lines. Ui = un-induced (i.e., not heat shocked). Hsp70 = heat shock 70 protein used to show the levels of proteins in the lanes of all western blots. Dl and DlΔI were detected with αDlEC. Da signals here (and the indicated signals elsewhere) were quantified relative to Hsp70 (western blots), rp49 (northern blots), or other indicated molecules, using the NIH Image 1.63 program. These experiments were repeated more than ten times. C . Western blots showing Da levels in the indicated cell mixtures, with (+) or without (-) Sca. These experiments were repeated five times. E(spl)C m3 expression appeared to be solely dependent on N activation and the Notch intracellular domain as it was promoted in S2-N cells treated with either S2-Dl cells or S2-DlΔI cells (Fig. 2A , lanes 7–12). As DlΔI lacks the intracellular domain, it is expected to behave only as a ligand of N and not generate any intracellular signal of its own in response to N binding. We observe comparable levels of SuH/N intra signaling with S2-Dl and S2-DlΔI cells (Fig. 2A , lanes 10, 12). This is not consistent with the in vivo findings that the Dl intracellular domain (lacking in DlΔI) is required for SuH/N intra signaling, possibly for promoting Dl internalization that results in exerting a 'pull' on N and increased production of N intra [ 21 , 25 - 29 ]. However, our results are consistent with other S2 cell studies showing that even fixed S2-Dl cells can promote production of SuH/N intra signaling in S2-N cells [ 19 ]. Thus, it is possible that that Dl internalization and pulling is not required for SuH/N intra signaling in S2 cells. In any case, in our S2 cell system, the S2-N and S2-Dl cells require shaking for formation of cell aggregates. As a consequence, we shake all cell mixtures, including those containing the secreted ligand Sca. This shaking might have simulated the pulling effect and overcome any deficiency DlΔI might have in this regard thereby resulting in a level of SuH/N intra signaling that is comparable to that produced by the full length Dl. We examined the expression of various proteins known to be involved in lateral inhibition to find out if Dl expression affected them. They were Numb, Dishevelled, Suppressor of Hairless, Wingless, Hairless, Hairy, Achaete, Da, and Armadillo. We found a relatively high level of Da protein in S2-Dl cells compared with the level in S2-DlΔI cells (Fig. 2B , lanes 1–2). Similar levels of Da were expressed in S2-DlΔI and S2 cells (data not shown). Two independently transfected S2-Dl cell lines also showed high levels of Da, and un-induced S2-Dl cells showed background levels of Da, indicating that Dl expression promotes Da expression (Fig. 2B , lanes 3–6). Increase in Da levels appeared to be specifically linked to Dl expression, as S2-N cells did not show an increase (Fig. 2C , compare lanes 1 & 3). Overall, Da expression in S2-Dl cells was 2.18X higher (+/- 0.37, p < 0.05) than the level in S2 cells, sometimes more than 5X higher. Here and in all cases to follow, the blots shown in the figures are the most representative blots among replications. Graphs show quantification, relative to standards or other proteins (as indicated), of signals on the blots composing the figures as the response can be assessed only in comparison to the control lanes in the same experiment. Pooling data from all replications of an experiment obscured the response, or misrepresented the data, due to variation between different batches of cells. Therefore, we computed error variance for the degree of response over all replications of an experiment. These values for important responses are mentioned in the text. The number of repetitions of an experiment is indicated in the figure legends. We examined Da levels in S2-Dl and S2-N cells that were treated or not treated with Sca conditioned medium. We found that Sca treatment decreased the levels of Da in S2-Dl cells (Fig. 2C , lanes 3–4). The levels in S2-N cells were low and unaffected by Sca treatment (Fig. 2C , lanes 1–2). These experiments suggested that Sca blocks accumulation of Da in S2-Dl cells (2.81X, +/- 0.59, p < 0.05). We also determined the levels of Da when S2-N and S2-Dl cells were together in the absence of Sca. The level of Da never increased (Fig. 2C , lanes 6–7). As N activation suppresses daughterless RNA expression [ 18 ], it was possible that N activation suppressed Da expression in S2-N cells and masked an increase in S2-Dl cells. To determine if this was the case, we compared the level of Da in mixtures of S2-DlΔI cells and S2 cells with mixtures of S2-DlΔI cells and S2-N cells. As S2-Dl and S2-DlΔI cells activate N equally well (see Fig. 2A , lanes 9–12), any change in Da level would be due to N activation. We found comparable levels of Da in the two samples (Fig 2C , lanes 8–9). Thus, S2-Dl cells do not appear to increase Da expression in response to S2-N cells. When S2-N and S2-Dl cells were together in the presence of Sca, the levels of Da protein and E(spl)C m3 RNA were very variable (data not shown). This was possibly due to the varying combinations of Sca effect on S2-Dl cells, Sca effect on S2-N cells, Dl effect on S2-N cells, and N effect on S2-Dl cells. Dl is processed to produce Dl intracellular domain (DlIC), constitutively, and the levels of DlIC increase upon N treatment [ 30 - 33 ]. Therefore, we examined the levels of DlIC following treatment of S2-Dl cells with S2-N cells or Sca medium. We found that the DlIC levels increased by 25 to 50% (relative to Dl levels) with both treatments (Fig. 3A and 3B ). We examined the levels of Da in S2 cells expressing DlIC and DlTMIC (lacking the extracellular domain only and including the transmembrane domain). The levels of Da in S2-DlIC and S2-DlTMIC were always comparable to or lower than the level in S2 or S2-DlΔI cells (Fig. 3C ). Also, we found Da levels to be negatively correlated with the accumulation of DlIC in time-course experiments (Fig. 3D ). This negative correlation could be a direct consequence of the accumulation of DlIC or due to autoregulation of the da gene [ 34 ]. We examined the levels of Da in flies expressing heat shock induced Dl, N, or Sca, in flies heterozygous for the null alleles of N or Dl, and in flies homozygous for a null allele of Sca. We found that Da expression was strongly associated with Dl expression rather than with N expression, and inconsistently associated with Sca expression. These data are consistent with our findings in S2 cells but are not shown, as we cannot clearly separate the effects of N, Dl, and Sca, the way we can do in S2 cells. The experiments described in this section indicate that Da accumulation is promoted by the full-length Dl, not by the Dl intracellular domains (DlIC or DlTMIC), and Sca suppresses the activity of the full length Dl. The experiments also indicate that Sca promotes E(spl)C m3 RNA expression in S2-N cells even in the absence of Dl. Figure 3 The levels of cleaved Dl intracellular domain is not associated with high levels of Da. A . Western blots (from a 8% SDS-PAGE) showing the level of Dl and DlIC in the indicated cell mixtures, with (+) or without (-) Sca. B . Western blots (from a 12% gel) showing the levels of Dl and DlIC in S2-Dl cells treated medium containing different levels of Sca. C . Western blots showing the levels of Da in the indicated cell mixtures. D . Western blots showing the levels of Da, Dl, and DlIC at different times following heat shock induction of Dl in S2-Dl cells. All experiments were repeated at least three times. Dl regulates expression of fringe and pangolin To gather additional evidence for Dl activity independent of its activity as a ligand of Notch, we performed microarray experiments using the Affymetrix Drosophila GeneChip Arrays to compare gene expression in S2 cells and S2-Dl cells. Many genes relevant to known Dl functions responded in S2-Dl cells (at p < 0.05, n = 3 × 2 pooled samples): axonal path finding genes (e.g., Gef64C, 39.38X,Up; Tenascin major, 6.77XUp), actin-based cell motility and kinases (Rho-Kinase, 15.08XUp; Rhophilin 3.4XUp; nemo 1.72XUp, basket 1.69XUp; pointed 2.2XUp), N signaling pathway genes (e.g., reaper, 2.26XUp; sanpodo, 1.91XUp), and oogenesis genes (e.g., swallow, 8.12XUp; sprouty, 3.58Xup). Expression of transformer , was also up (1.76X) and it is significant in the light of our observation that Dl promotes expression of Da: both Da and transformer are involved in sex determination. Expression of da RNA was not significantly increased in S2-Dl cells, possibly due to the negative part of the da gene autoregulation system [ 34 ]. The detailed analyses with validations will be published elsewhere. The experiment also identified fringe ( fng ) and pangolin ( pan ) as responding to Dl expression. fng is a glycosyl transferase that regulates the affinity of N for Dl [ 35 - 37 ], and possibly also the affinity of Dl for N [ 38 ]. pan is a transcription factor functioning in the Wingless (Wg) pathway [ 39 , 40 ]. Notch and Wg pathways interact closely at many differentiation events during development [ 24 , 41 - 44 ]. Therefore, we chose fng and pan for further investigation. Northern blot analyses showed that the expression of fng and pan was higher in S2-Dl cells compared with S2-N or S2-DlΔI cells (Fig. 4A ). DlIC and DlTMIC promoted expression of pan and fng weakly, if at all (Fig. 4B ). Two independently established S2-Dl cell lines also showed higher levels of fng and pan RNAs (Fig. 4C ). Sca treatment S2-Dl cells reduced the levels of fng and pan RNA (Fig. 4D ). This reduction was expected as Sca reduces the levels of the full length Dl (see Fig. 3 ). Thus, just as it was the case with Da expression, the full length Dl, not any of its parts, strongly promoted pan and fng expression. We examined the levels of fng and pan RNA in flies expressing heat shock induced Dl, N, or Sca, in flies heterozygous for the null alleles of N or Dl, and in flies homozygous for a null allele of Sca. We found that fng and pan RNA expression was strongly associated with Dl expression rather than with N expression, and inconsistently associated with Sca expression. These data are consistent with our S2-Dl cells data but are not shown, as we cannot clearly separate the effects of N, Dl, and Sca, the way we can do in S2 cells. Figure 4 Dl promotes expression of fng and pan . A . Northern blots showing fng and pan expression in the indicated cell mixtures at 0 and 45 minutes after cell mixing. B . Northern blots showing fng and pan expression in the indicated cell lines two hours after induction of expression. C . Northern blots showing fng and pan expression in two other independently established S2-Dl cell lines. Cells used for lanes 1–2 were uninduced (ui); cells used for lanes 3–4 were heat shock induced. D . Northern blots showing fng and pan expression in S2-Dl cells that were either untreated or treated with Sca medium. All experiments were repeated at least three times. The fng band marked with an asterisk corresponds to the published mRNA [35]. Only this band was used for fng quantification. The pan band shown is consistent with the information described in van de Wetering et al. [40] and Brunner et al. [39]. Discussion Our experiment addressed four questions. Does Sca bind Dl? If yes, does it affect any Dl activity? Are there Dl activities independent of its activity as a ligand of N? Is Sca capable of activating N in the absence of Dl? Results described in Figure 1 show that Sca binds Dl. This binding is not dependent on N as S2-Dl cells do not express N. We have previously shown that Sca binds N [ 14 ]. It is possible that Sca binds N or Dl stronger when they are present together on the same cell or on neighboring cells. It would be possible to test this in the future using Atomic Force Microscopy that is best suited for determining binding strengths of cell surface proteins like N or Dl [ 21 ]. Results in Figure 2A show that Sca can promote SuH/N intra signaling through N in the absence of Dl, as S2-N cells do not express Dl. However, numerous repetitions of the experiment indicate that Sca is not as potent as Dl in this regard. This is consistent with the fact that lateral inhibition is blocked in the absence of zygotic Dl, which does not affect proneural cluster formation and thereby Sca expression. It would have been relatively easy to determine if over-expression of Sca in the absence of Dl rescues SuH/N intra signaling phenotypes, and the extent of this rescue, if Dl did not have any activity independent of N. Hopefully, it would be possible in the future, when we better understand this Dl activity and are able to circumvent it. Results in Figure 2A also show that the expression of E(spl)C m3 gene, a target of SuH/N intra signaling pathway, is responsive only to N indicating that this pathway is unlikely to be involved in mediating Dl activities. Results described in Figures 2 , 3 , 4 and our microarray analysis show that Dl has activity independent of its activity as a ligand of N and Dl could be a receptor of Sca. This is clearly shown in experiments with S2-Dl cells that do not express N and we do not provide either N or Sca (Fig. 2B, C ; 4A ). The Dl activities we have described- promotion of expression of Da protein, fng RNA, and pan RNA- can be detected in vivo as well although the interpretation here is not simple due to the many possible interactions among N, Dl, and Sca that cannot be easily sorted out. However, these data (which we do not show) strongly suggest that the Dl activities we have described in S2 cells represent the in vivo Dl activities during development. The N independent Dl activity we have described is dependent on the full length Dl, not just on its intracellular domain or the extracellular domain (Figs. 2C ; 3 ; 5A-B). This is different from the situation with N whose activity is based on the activity of its intracellular domain [ 45 - 47 ]. Accordingly, treatment with Sca, which promotes production of the Dl intracellular domain, suppresses Dl activity related to Da rather than promote it (Figs. 2C ; 3 ). This observation also indicates that Sca is able to affect Dl activities. A clean dissection of Sca effects through N from its effects through Dl would require identification of Sca binding sites on Dl, and N and Dl binding sites on Sca. We know that Dl and Sca bind different regions of N [ 14 , 48 , 49 ]. It would not be too surprising if Dl bound N and Sca in different regions, and if Sca bound N and D in different regions. With that knowledge and suitable mutants, we might be able to determine whether N, Dl, and Sca activities function in a mutually exclusive or combinatorial manner in vivo . Dl activity that is independent of its N ligand activity has been speculated for some time. Efforts to identify it have intensified since the discovery that Dl gets proteolytically processed in the same manner as N [ 30 - 33 ]. However, it is extremely difficult to separate these two activities of Dl. The proof that the Dl activity we have identified actually functions during development in the expected manner, the details of the mechanisms underlying this function, and a better integration of the known functions of N, Dl, and Sca, will have to await more work which is neither quick nor simple. We hope that this work stimulates more efforts towards this task and makes this task a bit easier by identifying the potential of Sca as a regulator of Dl activity and the possibility that the full length Dl might be important for Dl activity independent of N, or Sca. Sca could also serve as a great tool for in vivo dissection of Dl response to N, as Sca and N appear to have a similar effect on Dl. It is quite likely that our experiments did not pick up Dl receptor activity in response to N or Sca. In any case, the potential developmental significance of our findings is briefly discussed below. Da is a widely expressed protein and cells requiring its function show only a modest increase in its levels [ 5 , 6 , 8 ] indicating that, just like N intra /SuH signaling, small changes in Da levels might be sufficient for initiating or augmenting NPC specification and promoting neuronal differentiation. Small changes in Da levels might also be imposed by the built-in autoregulation of the da gene [ 34 ]. According to the well-accepted lateral inhibition model in the field, Dl activity as a ligand of N is postulated to increase in the NPCs and N receptor activity in response to Dl is postulated to increase in the EPCs [ 11 ]. Accordingly, Dl expression has been observed to increase in the NPCs or their equivalent cell types in certain instances involving N and Dl functions [ 50 , 51 ]. Our data suggest that an increase in Da levels in these instances could be due to the accumulation of the full length Dl, not any its parts such as DlIC, DlΔI, or DlTMIC. The requirement for the intracellular and the extracellular domains to be linked might mean that we have detected Dl activity requiring Dl's presence at the membrane or in the cytoplasm. This is consistent with the report that the cellular transformation ability of Jagged 1, a mammalian Dl homolog, requires an intact protein containing both the extracellular and the intracellular domains [ 52 ]. It is possible that DlIC, in the nucleus [ 31 ], promotes other activity that is different from the one described here. It is also possible that Da, fng , or pan might not be the direct target of the full length Dl activity. Our microarray data indicate that many other genes (including some in the RAS or EGFR signaling pathways) are strongly up regulated in Dl expressing cells. It is possible that one of these genes is the primary target. It is also possible that Da, fng , or pan accumulation is significant only in the context of these other genes. We will have to await validation of other putative targets of Dl activity, and evaluation of their role in lateral inhibition or other activities involving N and/or Dl, to determine if Da, fng , or pan are typical or atypical targets of Dl activity. N/Dl binding and SuH/N intra signaling are strongly affected by the functions of glycosyl transferases such as fng . The possibility that Dl, and not N, regulates fng RNA expression might explain some of the very complex functions of these glycosyl transferases and the complex interactions between N and Dl during lateral inhibition. As N and Dl activities are known to strongly interact with the Wg signaling pathway, it is interesting that Dl promotes pan expression. It is possible that Dl activity independent of N accounts for some of the interactions between the N and the Wg pathways. So far, these interactions have been considered only from the perspective of N receptor activity. Lastly, our data suggest interesting interactions among Dl, N, and Sca in instances of lateral inhibition and tissue differentiation when their functions overlap. The full length Dl promotes Da accumulation, not any of its parts that might result from processing in response to N or Sca binding. Thus, both the processed N and Dl might promote EPC specification- processed N through E(spl)C RNA and processed Dl through suppression of Da expression. Consequently, lateral inhibition might initiate with symmetrical actions of N and Dl promoting EPC specification in all proneural cells. Sca might boost N and Dl processing in the incipient EPCs while suppressing them or not affecting them in the incipient NPCs. Thus, it is possible that Sca or Sca-like molecule have a role in breaking the symmetrical actions of N and Dl during certain lateral inhibition instances. It is also possible that Sca mediates long range N signaling during differentiation of some other tissues, either alone or in association with Dl, as proposed by Renaud and Simpson [ 13 ]. By extending our results, it might be possible to develop strong hypotheses for testing in vivo , cleanly sort the different activities of N, Dl, and Sca, and understand the fascinating in vivo developmental mechanisms involving N, Dl, and Sca. Conclusion Sca binds Dl and suppresses a Dl activity that is independent of Dl's activity as a ligand of N. This Dl activity requires the full length Dl and is not enhanced by expression of just the Dl intracellular domain, which is different from the mechanism underlying Notch activity. Da protein, fng RNA, and pan RNA responds positively to the N independent Dl activity we have discovered. These could be direct or indirect targets. Our microarray analysis has identified many more putative targets of N independent Dl activity that can be explored for a better understanding of the complex interactions among Dl, Sca, and N during Drosophila development. Methods DNA constructs Sca-Gfp: The stop codon of sca was replaced with a glycine codon and fused in-frame with GFP to obtain Sca-GFP. A Bam HI-KpnI fragment containing this sca sequence was cloned into pEGFP vector (Clontech). The XbaI fragment containing Sca-GFP coding fragment was cloned into the pCaSpeR-hs vector. DlΔI: A stop codon and a XbaI restriction site was introduced after the transmembrane domain using PCR. The PCR product was checked for mutations and used to replace the BstEII-BcgI fragment in the Dl cDNA. An Eco RI-XbaI fragment from this construct (Dl amino acid 1 to 620) was cloned into the pCaSpeR-hs vector. DlIC: The Dl intracellular region (codon 619 to the stop codon 881) was PCR amplified, checked for errors, and cloned into the BglII-XbaI sites in the pCaSpeR-hs vector. Cell lines and conditioned medium S2-N, S2-Dl, and S2-Sca cells have been previously described [ 14 , 20 , 49 ]. Other cell lines were established using the standard calcium phosphate transfection procedure and hygromycin selection. Conditioned medium was produced as described in Powell et al. [ 14 ], using serum-free or serum-containing Shields and Sang's M3 medium. For experiments, cells were heat shocked at 37°C for 30 minutes in a water bath, allowed to synthesize proteins for 2 hours, washed in culture medium without serum, mixed with the appropriate cell lines, and shaken gently in 14 ml falcon tubes for two hours or the indicated time. See Wesley and Mok [ 20 ] for more details. Immunoprecipitations, western blotting, northern blotting, RNA in situ, and protein staining Procedures described in Lieber et al. [ 43 ], Wesley [ 24 ], Wesley and Saez [ 18 ], and Wesley and Mok [ 20 ] were followed. Eight per cent SDS-PAGE systems were used for western blotting, unless otherwise indicated; 1% formaldehyde-MOPS agarose system for northern blottings. fringe cDNA (from Dr. Ken Irvine), rp49 cDNA, and rt-PCR amplified pangolin cDNA were used to prepare probes for northern blots. Incubation times with ligands were two hours for western blots and 45 minutes for all northern blots; it was three hours for fng and pan northern blot showing the effect of Sca (Fig. 4D ). Antibodies: αSca (mAb sca1) and αDlEC (C594.9B) were obtained from the Developmental Studies Hybridoma Bank; αGFP (G-6539) and αHsp70 (H-5147) from Sigma; αDlIC (GPC2) from Dr. Marc Muskavitch, αDlIC (dC-19) from Santa Cruz Biotechnology, αDa (DAM 109-10) from Dr. Claire Cronmiller; and αNI from Dr. Toby Lieber. Microarray analysis Heat shocked S2 and S2-Dl cells were treated with Sca or non-Sca medium for 45 minutes before extracting RNA. GeneChip Drosophila Genome Arrays from Affymetrix were used. RNAs were extracted, checked, and processed for hybridization according to procedures suggested by Affymetrix. We pooled RNA from two independent experiments and used three such pooled samples as replicates for each treatment. The MicroArray Core Facility at the University of Vermont prepared the probes, hybridized the chips, and statistically analyzed the data (using the GeneSifter program). We used the Microarray Suite program to examine the data. Authors' contributions LPM and TQ designed and carried out many of the experiments in cultured cells and flies; BB carried out the immuno-precipitation experiments with cultured cells and helped in interpretation of data; MLC made the Sca GFP construct and assisted in many experiments; AH performed some experiments in cultured cells and prepared and maintained cell lines; FA assisted in statistical analyses and interpretation of data; and CSW conceived the study, designed experiments, and performed or participated in many of experiments in cultured cells and flies. LPM, BB, and MLC helped CSW in drafting the manuscript. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555533.xml
535562	Density-independent population projection trajectories of chromosome-substituted lines resistant and susceptible to organophosphate insecticides in Drosophila melanogaster	Background Seasonal fluctuations in susceptibility to organophosphate insecticides were observed in the Katsunuma population of Drosophila melanogaster for two consecutive years; susceptibility to three organophosphates tended to increase in the fall. To examine the hypothesis that variation in fitness among resistant and susceptible genotypes could trigger the change of genetic constitution within the fall population, we investigated density-independent population projection trajectories starting from single adult females with characteristics of chromosome-substituted lines resistant and susceptible to the three organophosphates. Results Density-independent population projection trajectories, expressed as the ratios of the number of each chromosome-substituted line to that of line SSS, for which all chromosomes were derived from the susceptible line, showed significant declines in numbers with time for all the resistant chromosome-substituted lines. Conclusion The declining tendency in the density-independent population projection trajectories of the resistant chromosome-substituted lines could explain the simultaneous decline in the levels of resistance to the three organophosphates, observed in the Katsunuma population in the fall.	Background The development of insecticide resistance in insect pest populations is a population genetic process, in which insecticides select for initially rare resistant mutants within a population [ 1 ]. Although the development of insecticide resistance is an inevitable consequence of insecticide application, because the purpose of insecticide usage is to kill a certain portion of the insect pest population, insect populations that have developed resistance to insecticides sometimes exhibit reduction in levels of resistance to insecticides, after having been released from insecticide application. The cause of this reduction in resistance levels has been controversial [ 2 ]. Crow [ 1 ] said "Since the genes causing insecticide resistance were at low frequency in the population before the insecticide began to be applied, it must ordinarily be true that they are to some extent disadvantageous; otherwise they would have been common. Therefore the selection for resistance should ordinarily involve the replacement of the original genes with R factors that, in every respect except insecticide resistance, are deleterious from a survival standpoint." Under this theory, it could be expected that there is variation in fitness among resistant and susceptible genotypes, and that resistant genotypes have lower fitness than susceptible genotypes, which could result in the change of frequencies of resistance factors within a population. The Katsunuma population (Yamanashi Prefecture, Japan) of Drosophila melanogaster is one of the well-established natural populations in Japan [ 3 ]. Katsunuma is famous for its vineyards, which extend continuously through the town, and its wine production. In the fall, masses of squeezed grapes are dumped outside during the process of wine production, and Drosophila flies drastically increase in number on them [ 4 , 5 ]. In this natural population, we have observed seasonal fluctuations in levels of resistance to three organophosphates for two consecutive years; susceptibility to the three organophosphates tended to increase in the fall [ 5 ]. To reveal the genetic basis of the seasonal fluctuations in susceptibility to the three organophosphates, we have compared several fitness measures among chromosome-substituted lines, whose chromosomes were derived from a resistant or a susceptible line collected before the population started expanding [ 6 , 7 ]. Lower fitness measures were generally obtained for resistant chromosome-substituted lines, which supports the previous hypothesis that resistant genotypes have lower fitness than susceptible genotypes. In the Katsunuma population, the drastic change in food and breeding environments occurs from summer to fall. Almost density-independent conditions are suddenly created by masses of squeezed grapes. However, the period of these conditions might not be long enough for the population to attain a stable age-structure; rapid expansion and contraction occur during a rather short period of time. In fact, the Katsunuma population rapidly decreased in mid-November [ 7 ]. Therefore, it is also necessary to examine population trajectories during the phase before the population attained a stable age-structure. In this study, we further investigated density-independent population projection trajectories initiated from a single adult female for each chromosome-substituted line, after constructing a Leslie matrix for each line, which may reveal the responses of genotypes similar to each chromosome-substituted line to density-independent environments during a short period of time. Results Age-specific fecundity averaged over replications, which were used for constructing a Leslie matrix for each chromosome-substituted line, is shown in Figure 1 . Under the density-independent conditions in this study, a genotype similar to line SSS whose chromosomes were all derived from the susceptible line #451-4 could increase the number of individuals totaled over all age-classes to 1.0 × 10 7 on Day 50 and 3.6 × 10 13 on Day 100, starting from a single adult female (Fig. 2 ). Density-independent population projection trajectories for the other chromosome-substituted lines were expressed as the ratios of the total number of each line to that of line SSS. These are shown in Fig. 3 for resistant lines and in Fig. 4 for susceptible lines, respectively. Because a ratio of 1.0 (dotted lines in these figures) means that the number of individuals of the chromosome-substituted line is equal to that of line SSS, declining curves indicate that the relative number of individuals of that chromosome-substituted line is decreasing, compared to line SSS. All resistant lines showed decreasing ratios with fluctuations, compared to the total number of line SSS (Fig. 3 ). On Day 50, the ratio of the number of individuals to line SSS was 0.314 (0.848; 95 % upper confidence bound) for line RRR, 0.008 (0.066) for line RSR, 0.036 (0.142) for line SRR, and 0.181 (0.537) for line SSR. On Day 100, the ratio of the number of individuals to line SSS was 0.103 (0.736) for line RRR, 0.000 (0.005) for line RSR, 0.001 (0.014) for line SRR, and 0.022 (0.169) for line SSR. All susceptible lines also showed decreasing ratios with fluctuations, compared to line SSS, but the decline in the ratio was not significant in case of line RSS (Fig. 4 ). On Day 50, the ratio of the number of individuals to line SSS was 0.001 (0.002; 95 % upper confidence bound) for line RRS, 0.582 (1.345) for line RSS, and 0.018 (0.072) for line SRS. On Day 100, the ratio of the number of individuals to line SSS was 0.000 (0.000) for line RRS, 0.297 (1.479) for line RSS, and 0.000 (0.006) for line SRS. Figure 1 Age-specific fecundity for each chromosome-substituted line, averaged over replications. Each chromosome-substituted line is indicated by its chromosome composition (X, second and third chromosome in order). R and S indicate the origin of the chromosome: R for the resistant line #609-10 and S for the susceptible line #451-4. (solid line): resistant line; (dotted line): susceptible line. Figure 2 Density-independent population projection trajectories from a single adult female for line SSS. (red line): trajectory obtained from observed data; (black line): trajectory obtained from resampled data. Figure 3 Density-independent population projection trajectories, expressed as ratio to line SSS, for resistant chromosome-substituted lines. Each chromosome-substituted line is indicated by its chromosome composition (X, second and third chromosome in order). R and S indicate the origin of the chromosome: R for the resistant line #609-10 and S for the susceptible line #451-4. (red line): trajectory obtained from observed data; (blue line): 95% upper confidence bound for the true population projection trajectory; (black line): trajectory obtained from resampled data. Figure 4 Density-independent population projection trajectories, expressed as ratio to line SSS, for susceptible chromosome-substituted lines. Each chromosome-substituted line is indicated by its chromosome composition (X, second and third chromosome in order). R and S indicate the origin of the chromosome: R for the resistant line #609-10 and S for the susceptible line #451-4. (red line): trajectory obtained from observed data; (blue line): 95% upper confidence bound for the true population projection trajectory; (black line): trajectory obtained from resampled data. The distribution of 170 bootstrap replications of the log-transformed ratio of the total number of each line to that of line SSS on Day 100 is shown in Fig. 5 for resistant lines and in Fig. 6 for susceptible lines, respectively. These distributions seem reasonably normal. In fact, for each chromosome-substituted line, the log-transformed ratio obtained using the observed data, and mean and median of 170 bootstrap replications of the log-transformed ratio were very close (Table 1 ), suggesting that the log-transformed ratio obtained using the observed data was nearly unbiased for each chromosome-substituted line. Figure 5 170 bootstrap replications of the log-transformed ratio of the total number of each resistant chromosome-substituted line to that of line SSS at Day 100. Each chromosome-substituted line is indicated by its chromosome composition (X, second and third chromosome in order). R and S indicate the origin of the chromosome: R for the resistant line #609-10 and S for the susceptible line #451-4. Figure 6 170 bootstrap replications of the log-transformed ratio of the total number of each susceptible chromosome-substituted line to that of line SSS at Day 100. Each chromosome-substituted line is indicated by its chromosome composition (X, second and third chromosome in order). R and S indicate the origin of the chromosome: R for the resistant line #609-10 and S for the susceptible line #451-4. Table 1 log (ratio at Day 100) obtained from observed and resampled data. RRR RRS RSR RSS SRR SRS SSR Observed -0.987 -5.928 -4.103 -0.527 -2.941 -3.410 -1.652 Mean (Bootstrap replications) -0.967 -5.881 -4.156 -0.463 -3.025 -3.396 -1.685 Median (Bootstrap replications) -0.992 -5.872 -4.061 -0.511 -3.011 -3.378 -1.653 Each chromosome-substituted line is indicated by its chromosome composition (X, second and third chromosome in order). R and S indicate the origin of the chromosome: R for the resistant line #609-10 and S for the susceptible line #451-4. Discussion Population projection trajectories of chromosome-substituted lines Chromosome-substituted lines, whose third chromosomes were derived from the resistant line #609-10, showed resistance to organophosphates [ 6 ]. In D. melanogaster , one of the major mechanisms for organophosphate resistance is caused by mutated acetylcholinesterase, the target site of organophosphate insecticides [ 8 ]. Mutations in acetylcholinesterase can change the catalytic activity and stability of this enzyme, which may lead to a fitness cost associated with organophosphate resistance [ 9 ]. In this study, all the resistant chromosome-substituted lines showed a significant tendency towards a decrease in the ratio of the total number of individuals to that of line SSS, under density independent conditions (Fig. 3 ). This was especially so for resistant line SSR, for which only the third chromosome was derived from the resistant line #609-10 and the other two major chromosomes were from the susceptible line #451-4. These results suggest that resistant genotypes tend to decrease in frequency under the density-independent conditions, even during the phase before the population attains a stable age-structure. The calculations we have presented here do not, of course, relate to the changes in frequencies of genotypes in a sexually reproducing population. However, they should provide insight into whether resistance genes are likely to be prevented from spreading to fixation, since the initial rate of change in frequency of a rare gene in a randomly mating population will follow the dynamics that we have studied here ([ 10 ] pp. 149–166). In the previous study, we compared the intrinsic rate of increase among the chromosome-substituted lines used in this study [ 7 ]. Resistant lines generally showed lower intrinsic rates than line SSS, but line RRR, whose chromosomes were all derived from the resistant line, had the intrinsic rate of increase that was not significantly different from that of line SSS [ 7 ]. Although some interactions among resistant chromosomes were suggested, we could not determine the role of the interactions in the seasonal fluctuations in susceptibility to three organophosphates. Because the decline of line RRR in the ratio was relatively slow, compared to the other resistant lines (Fig. 3 ), interactions among resistant chromosomes may prohibit rapid decline in the relative number of individuals of resistant genotypes. However, these interactions cannot prevent the decline in frequency of resistant lines under density-independent conditions before the population attains a stable age-structure. Therefore, these results suggest that the interactions among resistant chromosomes may not play a significant role in the seasonal fluctuations in susceptibility to the organophosphates within the Katsunuma population of D. melanogaster . All the susceptible chromosome-substituted lines also showed a decline in this ratio with respect to line SSS, although the decline in line RSS was not significant (Fig. 4 ). Because these lines do not have resistance factor(s), these results obviously suggest that other factor(s), rather than the resistance factor(s), may affect the density-independent population projection trajectories of these lines during the phase before reaching a stable structure. Relatively high frequencies of fitness-related mutations have been maintained in the Katsunuma population of D. melanogaster (viability [ 3 , 4 ]; fertility [ 3 , 11 - 13 ]; productivity [ 14 ]). The frequency of recessive lethal genes on the second chromosome in 1997 was estimated as 25% and that of male sterile genes was 10%, with fluctuations since early 1960s [ 3 ]. Because resistance factors were not located on the second chromosome of the resistant line #609-10, the declining tendencies in lines RRS and SRS were not correlated with the resistance factor(s). Therefore, it is possible that the resistant line #609-10 accidentally possessed the above fitness-related mutation(s) in the Katsunuma population on the second chromosome. Resistant genotype frequency in the Katsunuma population Following the previous study, which introduced calculating the intrinsic rates of increase for the resistant and susceptible chromosome-substituted lines, this study sheds some more light on the genetic basis of the seasonal fluctuations in susceptibility to three organophosphate insecticides, observed within the Katsunuma population for two consecutive years [ 5 ]. Our results support the hypothesis that there is variation in fitness among resistant and susceptible genotypes. In particular, because the fitnesses of the resistant and susceptible chromosome-substituted lines were evaluated by the population projection trajectories generated by a single adult female during the phase before attaining a stable age-structure, which might represent a relatively short period of the density-independent conditions at Katsunuma, this study provide a further genetic basis for understanding the seasonal fluctuations in susceptibility to the three organophosphates from summer to fall. Variation in fitness among resistant and susceptible genotypes could change the frequencies of the resistant genotypes in the fall Katsunuma population, where almost ampler density-independent conditions are suddenly created by masses of squeezed grapes dumped outside. Because the third chromosome of the resistant lines confers resistance to all of the three organophosphates [ 6 ], a significant tendency towards a decline in the ratio of each resistant chromosome-substituted line relative to line SSS could explain the simultaneous decline in the levels of resistance to the three organophosphates, observed in the Katsunuma population [ 5 ]. Conclusions In this study, we calculated population projection trajectories of chromosome-substituted lines resistant and susceptible to three organophosphate insecticides. All the resistant lines showed a significant tendency toward decrease in the numbers relative to line SSS. This study strongly supports the hypothesis that there is variation in fitness among resistant and susceptible genotypes, and that resistant genotypes have lower fitnesses than susceptible ones. Methods Drosophila lines We obtained vital statistics from chromosome-substituted lines, whose chromosomes were derived from a resistant inbred line #609-10 and a susceptible inbred line #451-4, constructed by using a balancer stock, w ; Sp /SM1; Pr Dr /TM3 [ 7 , 15 ]. Both of the lines were derived from the same natural population collected at Katsunuma on July 31, 1997, when the D. melanogaster population had not yet started expanding [ 16 ]. The resistant line #609-10 had low-to-moderate levels of resistance to three organophosphates, malathion, prothiophos and fenitrothion [ 16 ]. Although at least two resistance factors, one on the second chromosome and the other on the third chromosome, were shown to be involved in the Katsunuma population of D. melanogaster [ 15 ], the resistance factor(s) for all the three organophosphates were located on the third chromosome in the resistant line #609-10 [ 6 ]. Flies used for experiments were grown on glucose-yeast-cornmeal-agar medium in glass vials (3 cm in diameter and 10.5 cm in height) in incubators at 25 ± 0.5°C with a photoperiod of 14: 10 (L: D) h. In this paper, each chromosome-substituted line was represented by its chromosome composition. For example, SRS indicates the chromosome-substituted line possessing the X and third chromosomes derived from the susceptible line #451-4 (S) and the second chromosome from the resistant line #609-10 (R). Population projection Density-independent population projection trajectories from a single adult female were obtained for each chromosome-substituted line, after constructing a 19 × 19 Leslie matrix for each line (Fig. 7 ; cf. [ 17 ]). Figure 7 A 19 × 19 Leslie matrix, used for calculating density-independent population projection trajectories from a single adult female. Age-specific fecundity, f ( x ), and survival probabilities, P ( x ), over 19 days were used to construct a 19 × 19 Leslie matrix for each chromosome-substituted line. A Leslie matrix consists of elements, which are age-specific fecundity (the first column in this matrix form, f ( x )), age-specific survival probabilities (the off-diagonal elements, P ( x )), and zero [ 10 ]. P ( x ) values lie between 0 and 1, whereas f ( x ) values are necessarily more than or equal to zero [ 18 ]. For age-specific fecundity f ( x ), some values are zero, depending on the reproductive schedule of the organisms concerned [ 18 ]. By multiplying a row vector n ( t ), whose components are numbers of each age-class, the number of each age-class at the next time unit, n ( t + 1), can be calculated as n ( t + 1) = n ( t ) L [ 10 ]. To construct a Leslie matrix, age-specific survival probabilities and fecundity were based on demographic data, collected for each individual adult fly (cf. [ 19 , 20 ]). These were the same data set used for calculating the intrinsic rate of increase for each chromosome-substituted line in the previous study [ 7 ]. One pair of adult flies that emerged within six hours was placed in a food vial and transferred into a new vial every day. When the flies were transferred, we checked whether the flies were alive or dead, and counted newly emerged adult flies every day. 16 pairs were prepared for each chromosome-substituted line. Half of the newly emerged adult flies on each day were considered in the estimates of age-specific fecundity in this study. Age-specific survival probability was assigned to be one when the transferred adult fly was alive, and zero when the fly was dead [ 19 , 20 ]. Because adult females were transferred, the age-specific survival probabilities from egg to pupal stages for the adult females were assigned to be one. Ages of the flies placed in the food vials were estimated as the duration from the entry of the adult flies to first emergence of their offspring. Because this length of immature stage varied among replications and among chromosome-substituted lines, ages of the adult flies were not necessarily assigned to be the same among them. Although the ages of adult females that produced no adult offspring cannot be assigned in this study, 14 out of the 16 females producing no adult offspring survived over the 19-day census period; therefore, age-specific survival probabilities for these females were assigned to be one. Two females producing no adult offspring, one RRS and one RSR female, died during the 19-day census period. These females were therefore excluded from the analysis, because the age-specific survival probabilities cannot be defined for these females. In the previous study, age-specific fecundity and survival probabilities over 19 days, based on each individual, were used for calculating the intrinsic rate of increase for each replication. In this study, the age-specific fecundity and survival probabilities over 19 days were used to construct a 19 × 19 Leslie matrix (Fig. 7 ), after averaged over replications. Density-independent population trajectories from a single adult female, which just started reproduction, were then projected, using the 19 × 19 Leslie matrix. Statistical procedures Density-independent population projection trajectories were estimated as the ratios of the number of each chromosome-substituted line, totaled over all age-classes, to that of line SSS for 100 days. 95% upper confidence bounds (one-sided; [ 21 ]) were set for the true population projection trajectories, after standard errors were obtained using the following bootstrap procedures [ 22 ]. For each bootstrap replication, 16 (15 for RRS and RSR) replications for each chromosome-substituted line were resampled with replacement from the observed data set, using a random number generator (Fortran 77 program). Age-specific fecundity and survival probabilities were averaged over resampled replications to construct a Leslie matrix for each line. After projecting the Leslie matrix for each line, the ratios of the total number of each line to that of line SSS for 100 days were estimated for each bootstrap replication. We collected 170 bootstrap replications for estimating standard errors for the ratios. Taking the distribution's shape into account, ratios were first log-transformed, and standard errors and 95 % upper confidence bounds were set for the log-transformed values. 95 % upper confidence bounds were then back-transformed into the normal scale. Authors' contributions TM carried out data collection and analyses, and drafted the manuscript. BC contributed to the computer programming and provided the theoretical background. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535562.xml
548288	Inflammatory peptides derived from adipose tissue	The low-grade inflammation seen with aging is noted particularly in subjects with the metabolic syndrome of aging. Insulin resistance, obesity/abdominal obesity, and risks for many age-related diseases characterize this common syndrome. It is becoming clear that this increased adipose tissue is not simply a reservoir for excess nutrients, but rather an active and dynamic organ capable of expressing several cytokines and other fat-derived peptides (FDP). Some, but not all, FDP may have a role in development of the metabolic syndrome but there is no evidence that these FDP are causing inflammation directly. We suggest that high levels of inflammatory peptides are markers for obesity/abdominal obesity seen with aging, but some may not necessarily have a causative role in the development of inflammation.	Because bone marrow and adipocytes are derived from the same stem cells, it is not surprising to find so many inflammatory peptides expressed in fat tissue. Many of these are classical inflammatory peptides derived from components of the adipose tissue and all are known also as fat derived peptides (FDP). With aging, there is a linear accumulation of adipose cells and percent of body fat increases. This increased body fat is characterized by increased visceral adiposity [ 1 ] and occurs despite the decreased subcutaneous fat and progressive sarcopenia typical of aging [ 2 ]. Visceral adiposity has been associated with greater risks for age-related diseases [ 3 ]. In addition, fat infiltration typical of aging occurs in many organs including liver and bone marrow. As adipose tissue accumulates throughout the body and in other organs, it is possible that this hyperplastic adipose tissue over expresses FDP. The metabolic syndrome is a common disorder consisting of a cluster of abnormalities including insulin resistance, dyslipidemia, and hypercoagulability and is associated with increased risk for cancer, Alzheimer's disease, type-II diabetes and atherosclerosis [ 4 ]. It is also associated with increased fat mass and increased inflammatory peptides. The obesity epidemic of the rapidly growing aging population makes understanding underlying relationship between adiposity, chronic inflammation and the metabolic syndrome essential. Increased inflammatory peptides are being studied as possible modifiable markers of the increased risk predictors of disease and possibly the underlying link between obesity and the poor clinical outcomes seen with the metabolic syndrome. More specifically, C-reactive protein (CRP) is the most well established inflammatory cytokine in the clinical setting but there are other inflammatory cytokines including IL-6, leptin, TNF- α , and other (non-cytokine) FDP, such as PAI-1, adiponectin and resistin, which may play a role in the pathogenesis, and/or serve as markers of risk in the metabolic syndrome. The fact that many of these peptides are derived from adipose tissue leads us to the question of whether adipose tissue itself is the underlying pathophysiological link between obesity and the poor clinical outcomes associated with the metabolic syndrome. We will provide a brief overview of some of the peptides associated with the metabolic syndrome. Cytokines with a potential role in the metabolic syndrome TNFα , leptin, and IL-6 are examples for cytokines that may have a role in the metabolic syndrome. TNF α , previously known as lymphotoxin and cachetin, is believed to be involved in the wasting that occurs during acute and chronic illness and malignancy. In the basal state TNF α is directly proportional to fat mass and has been shown to be involved in the development of insulin resistance [ 5 ]. In-vitro studies have demonstrated that TNF α decreases the insulin receptor tyrosine phosphorylation, and down regulates several steps in the insulin signaling pathway [ 6 - 9 ] while neutralizing agents for TNF α have been shown to improve insulin resistance. [ 10 ] Thus, TNFα is not only a classical cytokine but may be causal in the insulin resistance of the metabolic syndrome of aging . Leptin is a peptide derived from adipose tissue and like other cytokines acts through a cytokine receptor. It is expressed and secreted in direct proportion to fat mass. Leptin exerts is effect predominantly through receptors in the hypothalamus but it may also have peripheral actions [ 5 ]. Leptin serves as a marker of energy sufficiency by rapidly decreasing during starvation and weight loss. [ 11 ] With obesity, leptin levels are increased in proportion to fat mass, but its activity to decrease appetite seems reduced. Leptin appears to have an important role in energy regulation but no apparent role in development of inflammation . IL-6 is another cytokine derived from adipose tissue. Its expression and circulating levels correlate directly with obesity, and weight loss will lower circulating levels. Elevation of circulating IL6 is a predictor of the development of cardiovascular disease and diabetes [ 12 ]. Infusion of IL6 results in hyperlipidemia, hyperglycemia and insulin resistance in experimental models. [ 13 ] Additionally, IL6 decreases the expression adiponectin, an 'anti-diabetic' cytokine. [ 14 ] IL-6 plays a role in the development of insulin resistance and may directly cause induction of CRP . Other inflammatory cytokines such as IL-1 , IL-8, IL18, Serum Amyloid A, have been shown to be increased with obesity and may have a yet undetermined role in the syndrome. These cytokines are other examples of inflammatory markers which do not have a clear role in the causation of systemic inflammation. Non-cytokine Fat Derived Peptides with a role in the metabolic syndrome Adiponectin is highly expressed in adipose tissue, and is the one non-cytokine FDP that is protective from inflammation. Unlike most FDP, circulating levels are inversely proportional to obesity and therefore tend to be low in obesity. Adiponectin levels increase with weight loss and with use of insulin sensitizing drugs. [ 15 ] Adiponectin administration has been shown to improve insulin sensitivity. [ 16 ] Low levels of adiponectin have been linked to inflammatory arthrosclerosis in humans.[ 17 ] Animal models have shown that low adiponectin levels increase smooth muscle proliferation in response to injury, increase free fatty acids levels and cause insulin resistance.[ 18 ] The pro-diabetic and pro-atherogenic effects of low adiponectin levels seen in the metabolic syndrome provide a link between inflammation and obesity. Plasminogen activator inhibitor type-1 (PAI-1) is the primary inhibitor of fibrinolysis and is highly expressed in adipose tissue. Levels of PAI-1 are elevated in acute conditions such as deep venous thrombosis, and chronic conditions such as obesity, the metabolic syndrome of aging and diabetes. PAI-1 levels are correlated with adiposity and significantly overexpressed in the adipose tissue of obese compared to lean animals. [ 19 ] Levels are decreased by weight loss and drugs that improve insulin sensitivity [ 20 ]. The relationship of PAI-1 to obesity provides a potential link between the metabolic syndrome and hypercoagulabilty. Angiotesinogen (AGT) is a peptide that is produced in the liver and in adipose tissue. The strong correlation between obesity and hypertension implies that adipose tissue may play a role in blood pressure regulation and in fact there is a correlation between circulating AGT levels and obesity/hypertension [ 21 ]. Animal studies have shown that overexpression of AGT results in hypertension while under expression of AGT results in decreased blood pressures [ 22 ]. Resistin is a peptide which is elevated in obesity and appears to play a role in glucose homeostasis in rodents. In experimental models, resistin induces hepatic insulin resistance while anti-resistin antibodies have the opposite effect [ 23 ]. In humans, the role of resistin is less clear and it is not known what role it has glucose homeostasis or whether it directly relates to adipose tissue mass. The role of resistin in pathogenesis of inflammation is also unclear. Markers of inflammation or markers of obesity? Low grade inflammation is a predominant feature in the metabolic syndrome of aging and seems to be linked to the development of diabetes and poor vascular outcomes. We have briefly named several cytokines and other FDP that are generally increased with fat mass. Although many of these FDP have a role in metabolic homeostasis, many seem to lack distinct role in inflammatory pathogenesis. While many FDP have roles in vivo metabolism, we suggest that some levels of cytokines are increased because of the hyperplastic characteristic of adipose tissue, and their levels are better serve as marker of adipose tissue hypertrophy, rather than having a causal role in aging. Thus, whether aging is inflammatory state or whether it is a state associated with increased inflammatory marker is subject for further studies.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548288.xml
546229	Habitat characterization and spatial distribution of Anopheles sp. mosquito larvae in Dar es Salaam (Tanzania) during an extended dry period	Introduction By 2030, more than 50% of the African population will live in urban areas. Controlling malaria reduces the disease burden and further improves economic development. As a complement to treated nets and prompt access to treatment, measures targeted against the larval stage of Anopheles sp . mosquitoes are a promising strategy for urban areas. However, a precise knowledge of the geographic location and potentially of ecological characteristics of breeding sites is of major importance for such interventions. Methods In total 151 km 2 of central Dar es Salaam, the biggest city of Tanzania, were systematically searched for open mosquito breeding sites. Ecologic parameters, mosquito larvae density and geographic location were recorded for each site. Logistic regression analysis was used to determine the key ecological factors explaining the different densities of mosquito larvae. Results A total of 405 potential open breeding sites were examined. Large drains, swamps and puddles were associated with no or low Anopheles sp . larvae density. The probability of Anopheles sp . larvae to be present was reduced when water was identified as "turbid". Small breeding sites were more commonly colonized by Anopheles sp . larvae. Further, Anopheles gambiae s.l . larvae were found in highly organically polluted habitats. Conclusions Clear ecological characteristics of the breeding requirements of Anopheles sp . larvae could not be identified in this setting. Hence, every stagnant open water body, including very polluted ones, have to be considered as potential malaria vector breeding sites.	Background Urban malaria Urbanization is progressing fast worldwide. By the year 2030, more than 50% of the African population will live in urban areas [ 1 ]. It is anticipated that infectious disease problems including vector-borne diseases such as malaria might also increase in urban areas [ 2 ]. Keiser et al. [ 3 ] estimated that 24.8–103.2 million clinical malaria episodes occur annually in urban settings endemic for malaria. Urban malaria is generally characterized by: low transmission intensity, lack of immunity in the population and higher mortality rates in older age groups [ 4 ]. The distribution pattern of malaria transmission intensity is dependent on the degree of urbanization and on the distance from vector breeding sites [ 5 , 6 ]. Consequently, a high heterogeneity of transmission intensity is characteristic for urban malaria [ 7 ]. Increasing urban agriculture is thought to play a major role in increasing malaria in urban areas. However, this is not yet demonstrated, as it increases breeding sites for Anopheles sp . mosquito larvae, but it also raises the economic status of the population, allowing improved malaria protection [ 8 ]. Malaria is not only a personal health problem, but also an intolerable economic burden [ 9 ]. Investments in fighting malaria would result in substantial economic gains [ 10 ]. Therefore lowering the malaria burden in urban areas with a high economic development potential could result in improved economic development and serve as an example for other areas [ 7 ]. In urban areas, relatively few breeding sites are found per unit population [ 7 ]. In addition, the high human density usually allows a better access to curative and preventive health services. As a result, a number of malaria control options such as environmental control, including vegetation clearance, modification of river boundaries, draining swamps and insecticide application to open water bodies become possible and cost-effective [ 7 , 11 , 12 ]. With prompt access to antimalarials and other vector control interventions such as insecticide-treated bednets, integrated malaria control becomes realistic in African cities with a well developed municipal service delivery infrastructure [ 3 , 7 , 13 ]. Mapping of breeding sites and mosquito ecology as basis for integrated malaria control Every campaign to reduce malaria transmission aims to be as effective as possible given existing resources. Malaria control methods are affected by many setting-specific factors such as endemicity, vector species and behaviour, seasonality, disease patterns, health service factors and more. Since these factors are not distributed equally in space, accurate and timely information is required before malaria control can be planned and resources allocated properly. With regard to transmission reduction, attention must be paid to the areas of greatest vector abundance and precise risk maps of Anopheles sp . breeding sites are of major importance. Computerized maps have proven to be useful for this purpose, helping to understand malarial epidemiology and guiding interventions [ 14 - 18 ]. Besides geographic location, knowledge of ecological features of mosquito breeding sites is a potential key element for implementing efficient and effective larvae control measures. Such measures have been shown to be an important tool to reduce malaria endemicity [ 19 , 20 ]. In an urban environment Anopheles sp . mosquitoes adapt to new breeding sites created by urbanization, and hence their ecology might differ from rural settings [ 21 ]. Most African studies on Anopheles sp . mosquito larvae ecology have been conducted in rural settings [ 7 , 22 ], and findings from these studies might not be applicable to urban settings without adaptation. The spatial distribution of Anopheles sp . and Culicine larvae in open breeding sites in Dar es Salaam, Tanzania, were examined during an extended dry period and correlations between biological and physicochemical environmental factors and the abundance of mosquito larvae were determined. This work was carried out in order to collect baseline information for a larval control-based mosquito abatement programme implemented by the three municipalities of Dar es Salaam. Materials and Methods Study site Dar es Salaam is the biggest and economically most important city of Tanzania, with a population of more than three million people and an area of 1450 km 2 . The present study area covers 151 km 2 . in the central area of the city (Figure 1 ). Although highly urbanized, the study area is dotted with swamps, ponds and lakes and many additional water bodies are created for agricultural purposes. Combined with the hot and humid climate, this results in excellent breeding opportunities for Anopheles sp . mosquitoes. In Dar es Salaam the main malaria vectors are: Anopheles gambiae s.s ., Anopheles arabiensis , Anopheles funestus and Anopheles merus [ 23 - 25 ]. Figure 1 Breeding sites of Anopheles sp . larvae in Dar es Salaam, Tanzania. Study period The data collection was conducted from March 1 to May 29, 2003. Even though the study period was meant to be during the rainy season, the actual rainfall (236 mm) was less than half the long-term average from 1961–1990 (576 mm) [ 26 ]. Furthermore, the previous short periods of rainfall also failed, so this represents part of an extended drought period. Each area was visited only once in a cross-sectional survey of breeding sites. Mosquito identification To distinguish between A. gambiae s.l . ( gambiae s. s ., arabiensis , merus ) and A. funestus , larvae collected from the productive breeding sites were raised and hatched in the laboratory so that the adults could be identified morphologically by light microscopy using the key of Gillies and De Meillon [ 27 ]. Anopheles gambiae s.l . were not further identified to sibling species by PCR because such a methodology is not practical, affordable or particularly useful in the context of routine operations of a sustainable mosquito abatement program in Dar es Salaam. Breeding site identification and recording of ecological parameters Each ward within the study area was searched for open water bodies. To locate areas of potential breeding sites, the search was lead by municipal malaria experts with a good knowledge of the area. Furthermore, one-year-old geo-referenced digital aerial pictures (0.5 m ground resolution, produced by Geospace International, Pretoria, South Africa) were examined visually on the computer and all suspected open water areas were checked on the ground by extracting coordinates from the digital picture and locating the area in the field using a global positioning system (GPS) (eTrex © , Garmin International Inc., Olathe, US). In addition, 10 year-old breeding site maps from the Japan International Cooperation Agency (JICA) were consulted [ 24 ]. All open bodies of water were taken as potential breeding site and geo-referenced by GPS. The presence of larvae at high or low densities was determined by dipping. From every potential breeding site up to 10 dips were taken with a standard white 350 ml dipper. If Anopheles sp . could be seen without dipping or nearly every dip contained Anopheles sp . larvae, the site was defined as having a high Anopheles sp . density. Sites where only one or two dips out of 10 contained Anopheles sp . larvae were defined as having a low Anopheles sp . density. Sites where no Anopheles sp . larvae could be found in ten dips were recorded as empty. Pupae were not recorded as they cannot be differentiated from non- Anopheles species in the field. To attempt to quantify the relative mosquito larvae productivity of each breeding site, a productivity score was assigned to each breeding site according to its size and mosquito larvae density (Table 1 ). Sites which were dry at the time of visit were excluded. Table 1 Classification of larvae productivity in potential breeding sites Perimeter of site Perimeter < 1 m Perimeter 1–10 m Perimeter > 10 m Larval density Absent 0 0 0 Low 1 1 2 High 1 2 3 0 = No larvae productivity, 1 = Low larvae productivity, 2 = Medium larvae productivity 3 = High larvae productivity The same definition was also used to characterize Culicine mosquitoes (mainly consisting of the genera Culex , Aedes and Mansonia ) density in the same sites. To record habitat type, every site was categorized as one of the following: stream, small drain, large drain, swamp, rock pool, puddle, footprints, tyre track, artificial hole, concrete hole, artificial container and other. Further biological and physicochemical factors were measured, as shown in Table 2 . All visual classifications were done by the same person to maintain consistency. In addition, the general setting was qualitatively described, mentioning special features and general impressions. Table 2 Key parameters measured in the 327 sites that contained water, Dar es Salaam, Tanzania. Characteristics # % Characteristics # % Type of breeding site: Filamentous algae present 138 42.2 Stream 2 0.6 Large drain 29 8.9 Single-celled algae present 18 5.5 Small drain 16 4.9 Swamp 92 28.1 Predators present 118 36.1 Rock pool 0 0 Puddle 22 6.7 Shade Foot/hoof print 4 1.2 0–25% 250 76.5 Tyretrack 1 0.3 26–50% 58 17.7 Artificial hole 82 25.1 51–75% 16 4.9 Concrete hole 9 2.8 75–100% 3 0.9 Artificial container 0 0 Other 69 21.4 Maximum depth more than 0.5 m 108 32.7 General surrounding Size High density housing 236 71.6 Perimeter < 1m 17 5.2 Fields 57 18.0 Perimeter 1–10 m 128 39.4 Industry 19 5.8 Perimeter > 10 m 182 55.4 Other 15 4.6 Turbidity Distance to nearest inhabited house Clear 137 41.9 <10 m 179 54.7 Turbid 160 48.9 10–100 m 127 38.8 Very turbid 30 9.2 >100 m 21 6.4 Salinity mS/cm (rounded to full numbers) Distance to nearest potential resting site 0–1 138 42.3 <10 m 327 100 2–3 145 44.5 10–100 m 0 0 4–5 22 6.7 >100 m 0 0 6–7 4 1.2 8–9 3 0.9 Anopheles larvae density 10–14 4 1.2 Absent 125 38.2 15–19 2 0.6 Low density 64 19.3 >20 8 2.5 High density 138 42.5 Temperature °C (rounded to full numbers) Culicine larvae density 25–26 9 2.8 Absent 139 43.1 27–28 34 10.4 Low density 43 13.1 29–30 92 28.2 High density 145 43.7 31–32 71 21.7 33–34 63 19.3 Anopheles and Culicine pupae density 35–36 39 12.0 Absent 247 75.5 37–38 12 3.7 Low density 37 11.3 39–40 5 1.5 High density 43 13.1 pH (rounded to full numbers) Grass in the middle of site present 134 41.0 6 3 0.9 7 111 34.1 Grass along the edges present 244 74.6 8 169 52.0 9 46 12.3 Floating plants present 65 19.9 10 2 0.6 Floating algae present 41 12.5 Statistical analysis Statistical analysis was carried out with SPSS V 11.0 (SPSS Inc., Chicago, US). Logistic regression analysis was used to determine the importance of key factors for explaining the different mosquito larvae densities. For each group of mosquitoes (Anopheles sp . and Culicines) two logistic regression models were fitted: one model was used to determine the key factors influencing whether larvae were present or absent, and one model was used to determine the key factors influencing whether larvae were present at low or high density. For the second model only data from sites that contained at least one larva were used. All measured parameters were included in the logistic models. The three categories "stream", "foot/hoofprint" and "tyre track" were included in the baseline category "other" as they had only four or less records. No "artificial containers" or "rockpools" were found, consequently these categories are not presented in the analysis. Salinity, temperature and pH were measured with an electronic device (HANNA © HI 98130 Combo pH&EC, Hanna Instruments Inc., Kehl am Rhein, Germany). Conductivity measured in milliSiemens/cm was the indicator to measure salinity. The continuous variables "salinity" "temperature" and "pH" were categorized into quartiles for ease of interpretation. As this study is focused on Anopheles sp . larvae, only the significant parameters are presented for the Culicine models. To reduce the risk of α errors given the large number of variables that where investigated, only variables with p-values below 0.01, or those showing a consistent trend in both regression models with p-values below 0.05, were considered in the discussion. Ethics This study did not involve human subjects and permission was obtained from the National Institute of Medical Research in Tanzania (NIMR/HQ/R.8a/Vol.lX/l 12, No. 2003-110-CC-2003-63). Results Open breeding sites in Dar es Salaam A total of 405 potential mosquito breeding sites were examined and mapped. 19% were dry at the time of visit. Of the 327 sites that contained water, 62% were productive for Anopheles sp . and 57% were productive for Culicine mosquitoes. From all 150 adult mosquitoes reared from the larvae samples, only A. gambiae s.l . could be found. In one productive breeding site salinity was above 1%, suggesting that these larvae were Anopheles merus [ 28 ]. The 202 sites productive for Anopheles sp . were made up of 69% low larvae density sites and 31% high larvae density sites. Of the 186 sites productive for Culicine larvae 23% had low larvae density and 77% had high larvae density. Breeding sites of Anopheles sp. and Culicines and their productivity are shown in Figures 1 and 2 . Figure 2 Breeding sites of Culicine larvae in Dar es Salaam, Tanzania. Key factors determining the presence/absence or low/high density of mosquito larvae The frequencies of the different parameters are shown in Table 2 . Presence/absence of Anopheles sp. larvae (Table 3 ): Large drains, swamps and puddles were much less likely to contain Anopheles sp . larvae than other habitats. Very turbid water diminished the chance that Anopheles sp . larvae were present. Breeding sites with a size of less than one meter were more likely to contain Anopheles sp . larvae. Table 3 Output of logistic regression model with presence versus absence of Anopheles sp . larvae as outcome and ecological parameters as explanatory variables. Variable Odds Ratio 95% Confidence Interval for OR P-Value Lower Upper Type of breeding site Other 1 Large drain .145 .046 .459 .001 Small drain .492 .111 2.188 .352 Swamp .139 .055 .346 .000 Puddle .196 .051 .752 .018* Artificial hole .481 .186 1.245 .132 Concrete hole 1.407 .156 12.702 .761 General surrounding High density housing 1 .118 Other .812 .210 3.131 .762 Industry .211 .055 .808 .023* Fields .502 .207 1.216 .127 Distance to nearest inhabited house <10 m 1 10–100 m 1.889 .473 7.541 .368 >100 m 1.839 .940 3.596 .075 Culicine density high 1 low .856 .369 1.988 .718 absent .687 .356 1.328 .265 Size of site Perimeter > 10 m 1 Perimeter 1–10 m 1.932 .928 4.020 .078 Perimeter < 1 m 22.825 2.094 248.843 .009** Turbidity of water clear 1 turbid .469 .231 .954 .037* very turbid .167 .059 .473 .001** Salinity measured by conductivity [mS/cm] (Quartiles) 2.6–20.0 1 1.7–2.5 .522 .228 1.195 .124 1.1–1.6 1.418 .576 3.490 .447 0.1–1.0 .728 .313 1.696 .462 Temperature (°C) (Quartiles) 25.1–29.2 1 29.3–31.3 1.758 .676 4.571 .247 31.4–33.4 .731 .306 1.744 .480 33.5–40.0 1.148 .507 2.597 .741 pH of water (Quartiles) 8.1–10.1 1 7.7–8.0 1.773 .711 4.426 .220 7.4–7.6 .825 .330 2.059 .680 6.1–7.3 .439 .180 1.070 .070 Predators present 1.931 1.031 3.619 .040* Sun-lit (less than 50% of surface shaded) 1.913 .575 6.363 .290 Depth >0.5 m .750 .393 1.434 .384 Grass present .952 .601 1.508 .834 Floating vegetation present .935 .570 1.534 .790 Submersed vegetation present 1.201 .679 2.124 .529 * = p < 0.05 ** = p < 0.01 High/low density of Anopheles sp. larvae (Table 4 ): When Anopheles sp . larvae were present in large drains, swamps and puddles, these sites were much less likely to contain high densities of Anopheles sp . larvae. Table 4 Output of logistic regression model with high versus low Anopheles sp . larvaedensity as outcome and ecological parameters as explanatory variable. Variable Odds Ratio 95% Confidence Interval for OR P-Value Lower Upper Type of breeding site other 1 large drain .124 .021 .739 .022* small drain .768 .109 5.389 .791 swamp .182 .053 .629 .007** puddle .165 .033 .832 .029* artificial hole .712 .198 2.556 .602 concrete hole .348 .032 3.727 .383 General surrounding high density housing 1 other .358 .056 2.290 .278 industry 2.675 .326 21.950 .359 fields .925 .275 3.112 .899 Distance to nearest inhabited house <10 m 1 10–100 m .658 .261 1.656 .374 >100 m .409 .054 3.069 .384 Cuttcine density high density 1 low density .728 .221 2.396 .602 absent 1.078 .432 2.688 .872 Size of site perimeter > 10 m 1 perimeter 1–10 m 1.565 .576 4.255 .380 perimeter < 1 m .907 .166 4.950 .911 Turbidity of water clear water 1 turbid water 1.653 .692 3.951 .258 very turbid water 2.116 .372 12.030 .398 Salinity measured by conductivity [mS/cm] (Quartiles) 2.7–20.0 1 1.7–2.6 1.638 .479 5.599 .431 1.2–1.6 .818 .251 2.671 .740 0.1–1.1 .687 .227 2.079 .507 Temperature (°C)(Quartiles) 34.1–40.0 1 31.6–34.0 1.004 .315 3.203 .994 29.4–31.5 1.312 .356 4.830 .683 25.1–29.3 .619 .176 2.184 .456 pH of water (Quartiles) 8.2–10.1 1 7.8–8.1 .882 .228 3.419 .856 7.5–7.7 .777 .200 3.027 .716 6.5–7.4 .793 .198 3.182 .744 Predators present 1.322 .530 3.302 .550 Sun-lit (less than 50% of surface shaded) 1.035 .184 5.832 .969 Depth >0.5 m .460 .174 1.219 .118 Grass present .801 .272 2.362 .688 Floating vegetation present .352 .130 .952 .040* Submersed vegetation present 1.745 .725 4.197 .214 * = p < 0.05 ** = p < 0.01 Presence/absence of Culicine larvae (Table 5a ): Turbid water was clearly associated with the presence of Culicines. Breeding sites with pH values of 7.6 or less were also more likely to contain Culicine larvae. Table 5 Output from a logistic regression model with (a) presence versus absence of Culicine and (b) high versus low density of Culicine larvae as outcome, and ecological parameters as explanatory variable. The same variables as in tables 2 and 3 were included in the model, but only significant results are shown. 5a Variable Odds Ratio 95% Confidence Interval for OR P-Value Lower Upper General Surrounding High density housing 1 Other .646 .170 2.453 .520 Industry .177 .041 .771 .021* Fields .692 .302 1.585 .384 Turbidity of water Clear 1 Turbid 3.620 1.872 7.000 .000** Very turbid 2.689 .981 7.369 .054 pH of water (Quartiles) 8.1–10.1 1 7.7–8.0 1.986 .845 4.669 .116 7.4–7.6 2.916 1.252 6.792 .013* 6.1–7.3 2.586 1.109 6.030 .028* 5b Variable Odds Ratio 95% Confidence Interval for OR P-Value Lower Upper Type of breeding site Other 1 Large drain 4.207 .575 30.801 .157 Small drain .836 .054 12.894 .898 Swamp 1.811 .507 6.467 .361 Puddle 2.797 .345 22.650 .335 Artificial hole 1.137 .288 4.486 .855 Concrete hole .059 .005 .733 .028* pH of water (Quartiles) 8.0–9.0 1 7.6–7.9 2.291 .748 7.014 .146 7.4–7.5 2.485 .707 8.740 .156 6.4–7.3 4.024 1.230 13.158 .021* High/low density of Culicine larvae (Table 5b ): A pH value of less than 7.3 was associated with high Culicine larvae density. Productive Anopheles sp . gambiae s.l . mosquito breeding sites in Dar es Salaam: unexpected results Contrary to the conventional thinking that A. gambiae s.l . only breeds in rather clean and clear water [ 28 , 29 ], such larvae were found in habitats organically polluted by rotting vegetation, human faeces or oil. Such sites were the drain of an oil refinery, one organically polluted swamp used as garbage dumping area and one sewage pond with organic pollution from human faeces (Figures 3 and 4 ). Except for the breeding site type "tyretrack", for which only one potential habitat was identified, An. gambiae s.l . could be found in all types of habitats recorded during the study. Figure 3 Sewage pond in Temeke municipality, Dar es Salaam, Tanzania. Figure 4 Dipper content from sewage pond in Temeke municipality (Figure 3). Socio- and geo-ecological environments with high mosquito breeding site density In our study six environments were defined where larval control interventions are of high priority due to the large number of highly productive breeding sites for Anopheles sp . Most environments identified as potentially suitable for mosquito breeding in Dar es Salaam could be described by a close interaction between geo-ecological settings and the influence of humans. Slopes on riverbeds, riverbeds, borders of swamps, stagnant drains and rivers, areas with restricted access and sites along railway lines made up the six high productivity environments (for further details see: Sattler [ 30 ]). As Dar es Salaam is highly populated, houses have been built nearly everywhere. Only swampy areas, riverbeds and steep slopes are not, or only sparsely, populated due to the danger of flooding or landslides. Further, housing is forbidden on military property or other restricted areas such as the international airport. All areas defined as potentially productive have a very high water table, as found in most parts of Dar es Salaam. As a result, these areas represent also suitable land for small-scale farming. The type of agriculture using " matuta " is most likely to lead to mosquito breeding. Matuta is a type of agriculture where plants are grown on top of little ridges, while between the ridges deep furrows are dug. These furrows are often filled with a little water, forming shallow pools perfectly suitable for Anopheles sp . breeding (Figure 5 ). Further, rice fields, shallow wells and irrigation channels were also found to be very productive for Anopheles sp . in the focus areas. Figure 5 "Matuta" type of agriculture in Dar es Salaam, Tanzania. Discussion Main limitations of the study This study represents a snapshot of a highly dynamic system and presents the first results of a larger malaria control program. More intensive longitudinal studies are currently being undertaken to complement these results. The study was implemented during an exceptionally dry year and, although the study took place during the rainy season, it actually rained very little [ 26 ]. As a result, it is unclear whether the breeding site structure found during the three months of our sampling period was representative of the structure found in years with normal rainfall. Similarly, the measured ecological parameters might be different during more normal years, as the selectivity of mosquitoes for oviposition sites can be greatly diminished during dry periods because of the limited availability of aquatic habitats [ 27 ]. In years with normal rainfall, it is very likely that many habitats that contained larvae during the study period would be unproductive due to a flushing effect, while other small productive sites might appear. However, for the purpose of reducing mosquito density by larvae abatement, the time when the mosquito population is most vulnerable is the dry period. A. gambiae s.s ., A. arabiensis and A. funestus survive during the dry period in discreet habitats, making them an easier target for control interventions [ 31 ]. Dry season larval control is for example the rule in South Africa [ 32 ]. Very small breeding sites could not be detected by aerial pictures and were, therefore, largely excluded from the study. This could result in the omission of factors potentially important for mosquito control interventions. Further, mosquitoes do not necessarily lay eggs every day in each potential breeding site, thus the reason why a site did not contain larvae could simply be because no eggs were deposited within the last week. One of the key objectives of the study was to detect ecological factors determining Anopheles sp . larvae productivity. But within a city, the influence of humans can never be neglected. The pollution from waste such as oil, soap or industrial by-products is important in potential breeding sites in Dar es Salaam. Larvae may have been absent due to pollution by elements that could not be detected in the frame of this study. Anopheles sp . mosquitoes were not classified down to species level. The goal of the study was to characterize important breeding sites of Anopheles sp . mosquitoes and, consequently, potential foci of malaria transmission, regardless of the species. This is because in the context of sustainable operations in a routine mosquito abatement programme, municipal staff cannot be expected to identify all Anopheles larvae samples to species level without rendering sampling procedures prohibitively laborious and expensive. To achieve a satisfactory impact, exhaustive targeting of all potential vector species is necessary anyway. Furthermore, community acceptance of vector control programmes in Dar es Salaam has been shown to require suppression of all mosquito species, rather than only malaria vectors [ 25 ]. Ecological factors influencing mosquito larvae density The abundance of Anopheles sp . and Culicine larvae seems to be influenced differentially by ecological parameters, as none of them had a significant effect in all four regression models. Only one factor influenced the presence or absence of both Anopheles sp . and Culicine larvae. In turbid breeding sites Culicine larvae were much more likely to be present, whereas Anopheles sp . larvae were much more likely to be absent. Bates [ 29 ] supports this finding of Anopheles sp . breeding in rather clear water, but Gimnig et al . [ 33 ] found increasing A. gambiae s.l . larvae densities with increasing turbidity. Robert et al . [ 34 ] found a clear water preference by A. ambiensis breeding in wells in urban Dakar. A study by Ye-Ebiyo et al . [ 35 ] found that the production of A. arabiensis was favoured in moderately turbid water, while excessive turbidity limited the production of larvae. The proximity to flowering maize with pollen as food source compensated for the development failure induced by excessive turbidity. Clearly, the simple definition of "turbidity" might not be precise enough. Water which is turbid from particles not edible for Anopheles sp . larvae could disfavour the production of larvae, while water turbid from food particles represents a very suitable habitat. The preference of Culicine mosquitoes for turbid water is coherent with their known breeding site preferences, as they breed successfully in rather polluted environments such as blocked drains and septic tanks [ 36 , 37 ]. Key factors influencing the density of Anopheles sp . larvae In small breeding sites with diameters less than 1 m, Anopheles sp . were more likely to be present than in larger habitats. This is consistent with the general description of breeding sites for A. gambiae s.l ., but not with those for A. funestus [ 27 , 36 ]. However, this finding could be biased by the fact that rain was lacking for several weeks, evaporating the bigger pools and concentrating the larvae, hence making them easier to detect. Further, larval density of small breeding sites might be increased due to a higher sampling intensity per unit area. Anopheles larvae were less likely to be present in swamps, and when so, they were found in low densities. This finding matches the known preference of A. gambiae s.l . for temporary sites [ 36 , 38 ]. Even though swamps were less likely to harbour Anopheles sp ., the importance of these habitats should not be underestimated because of their great size and their role in supplying water for irrigation ditches, rice fields and various agricultural activities. "Puddle" as a type of breeding site was not favoured by A. gambiae s.l . larvae, and when Anopheles sp . larvae were present there, they were likely to be present in low densities. This finding is not consistent with established habitat descriptions for Anopheles sp . larvae [ 25 , 39 ]. Large drains were likely to have low Anopheles sp . densities. This result agrees with findings by Yamagata [ 24 ]. Large drains were often organically polluted with waste water and, therefore, less suitable for Anopheles sp . breeding [ 21 , 28 , 38 ]. Key factors only influencing the density of Culicine larvae A pH value of less than 7.3 favoured high Culicine densities and a value of less than 7.6 favoured their presence. These results showed clearly that Culicine larvae favoured a pH-neutral environment. Anopheles sp . breeding sites: unexpected findings The findings of this study revealed that A. gambiae s.l . was found in a sewage pond (Figure 3 and 4 ) and in one swamp extremely polluted with organic matter. These findings from Dar es Salaam, together with other studies, could indicate a change of Anopheles sp . breeding requirements in urban settings. In Lahore, Pakistan, Anopheles sp . mosquitoes were found in the waste water system [ 40 ]. In Accra, Ghana, data collections since 1911 indicate that A. gambiae s.l . adapted to breeding in organically polluted water habitats [ 21 ]. As most studies of Anopheles sp . larval ecology have been conducted in rural settings, it is likely that unpolluted breeding sites are found and described far more often [ 7 , 22 ], giving a biased impression. One of the main problems is the term "polluted habitat", as it has never been clearly defined. The findings of this study show that Anopheles sp . larvae can breed in nearly every kind of water accumulation. For every ecological factor identified as enhancing or reducing Anopheles sp . larvae productivity, at least one breeding site was found that contradicted these findings. Hence, all water bodies in an urban environment should be considered as potential breeding places and a target for larval control. Defined socio- and geo-ecological environments with high mosquito breeding sites density Slopes to riverbeds, riverbeds, borders of swamps, stagnant drains and rivers, areas with restricted access and areas along railway lines represented environments where most breeding sites were found in Dar es Salaam. These sites where mostly associated with agriculture activities. Afrane et al . [ 8 ] stated that areas of high mosquito density tended to follow valleys, where breeding sites were most persistent. Also, in Brazaville the valleys with vegetable gardens and crops were identified as some of the most suitable places for Anopheles sp . breeding [ 5 ]. In Dakar, one big marshy area was responsible for the production of nearly all adult Anopheles sp . mosquitoes within a distance of one kilometer [ 6 ]. Similar results were found by Staedke et al . [ 41 ]. Ponds close to the embankments of a railway line are presented as potential dry-season refugia for mosquito by Charlwood et al . [ 31 ]. The increasing urbanization of Dar es Salaam will probably help to reduce risk areas in the long-term, as open spaces get rare and swampy areas are filled to regain building land, but unplanned city growth along the city edges could increase the number of breeding sites [ 22 ]. In Brazaville, areas with the lowest malaria transmission intensity corresponded to the oldest and most densely populated districts [ 5 ]. Furthermore, high density housing has been shown to reduce breeding sites more than medium-density housing in Kisumu and Malindi, Kenya [ 22 ]. Conclusions Mapping of malaria risk on the basis of breeding sites plays an important role for urban malaria control programs. Also, initial risk mapping of breeding sites, combined with improved knowledge of mosquito ecology and their interactions with humans, is crucial to understand the epidemiology of urban malaria. The present study, in accordance with other studies of Anopheles sp . on urban larval ecology, showed that Anopheles sp . mosquito larvae are not restricted to clearly defined habitats. Therefore, malaria control interventions, such as environmental measures or insecticide application, have to consider all open water bodies as potential breeding sites. Specific malaria control interventions are currently developed and tested by the three municipalities of Dar es Salaam. Authors' contributions MS designed and implemented the study, analyzed the data and drafted the manuscript. DM, MK and ZP were involved in designing and implementing the study and analyzing its data. MT participated in the study design, data analysis and drafting of the manuscript. GK and CL assisted in the study design and implementation, data analysis and writing of the manuscript. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546229.xml
539358	An informatics search for the low-molecular weight chromium-binding peptide	Background The amino acid composition of a low molecular weight chromium binding peptide (LMWCr), isolated from bovine liver, is reportedly E:G:C:D::4:2:2:2, though its sequence has not been discovered. There is some controversy surrounding the exact biochemical forms and the action of Cr(III) in biological systems; the topic has been the subject of many experimental reports and continues to be investigated. Clarification of Cr-protein interactions will further understanding Cr(III) biochemistry and provide a basis for novel therapies based on metallocomplexes or small molecules. Results A genomic search of the non-redundant database for all possible decapeptides of the reported composition yields three exact matches, EDGEECDCGE, DGEECDCGEE and CEGGCEEDDE. The first two sequences are found in ADAM 19 (A Disintegrin and Metalloproteinase domain 19) proteins in man and mouse; the last is found in a protein kinase in rice ( Oryza sativa ). A broader search for pentameric sequences (and assuming a disulfide dimer) corresponding to the stoichiometric ratio E:D:G:C::2:1:1:1, within the set of human proteins and the set of proteins in, or related to, the insulin signaling pathway, yields a match at an acidic region in the α-subunit of the insulin receptor (-EECGD-, residues 175–184). A synthetic peptide derived from this sequence binds chromium(III) and forms a metal-peptide complex that has properties matching those reported for isolated LMWCr and Cr(III)-containing peptide fractions. Conclusion The search for an acidic decameric sequence indicates that LMWCr may not be a contiguous sequence. The identification of a distinct pentameric sequence in a significant insulin-signaling pathway protein suggests a possible identity for the LMWCr peptide. This identification clarifies directions for further investigation of LMWCr peptide fractions, chromium bio-coordination chemistry and a possible role in the insulin signaling pathway. Implications for models of chromium action in the insulin-signaling pathway are discussed.	Background Type II diabetes continues to grow as a worldwide epidemic; it is expected that this disease will surpass 300 million cases by 2025 [ 1 - 5 ]. Understanding the complex signaling mechanisms of insulin receptor and downstream factors, e.g. IRS1 and IRS2 [ 6 ], is crucial in the design of effective therapeutic strategies. Clarification of such signaling mechanisms is expected to lead to discoveries to cure or prevent diabetes and other metabolic conditions [ 1 ]. Current strategies include the search for small synthetic or naturally-derived molecules to act upstream of IRS1, to adequately compensate for insulin dysregulation [ 7 , 8 ]. One unknown facet in this complex problem is the role that chromium may play in the regulation of glucose metabolism; the molecular basis of chromium action in biological systems has not been definitively explained [ 9 , 10 ]. While the toxicology Cr(VI) has been well characterized [ 11 ], an understanding of the biochemistry and action of Cr(III) continues to elude researchers within this field [ 9 , 11 - 18 ]. The lack of good mechanistic models and experiments currently limits researchers' ability to assess the relative importance of Cr(III) and its possible roles in glucose metabolism, obesity or Type II diabetes. Recently, a Cr-peptide fraction isolated from bovine liver [ 19 - 23 ] was shown to potentiate the action of insulin. The amino acid composition of this "low molecular weight Cr" complex (LMWCr, containing 3–4 chromic atoms) was identified to be approximately E:G:C:D::4:2:2:2 [ 18 , 21 , 24 ], though the sequence remains unknown (Figure 1 ) and there appears to be some difficulty in exactly repeating this work. Although not specifically related to Cr(III) metabolism, Ramasami and co-workers report that Cr(III) induces structural changes and long range ordering in collagenous tissues and suggest the formation of multinuclear Cr-oxo and hydroxo clusters (2, 3, or 4 Cr atoms) holding the proteinaceous molecular assembly together [ 25 , 26 ]. In this study, we report on a bioinformatics search for the LMWCr peptide, and propose that the peptide that binds to Cr(III) may be the pentameric sequence (-EECGD-) as it is found within the insulin receptor (INSR; Swiss-Prot: P06213) (-NKDDN EECGD -, residues 175–184) and conforms to the predicted stoichiometry. Further, visible absorption data and electron paramagnetic measurements of the synthesized disulfide linked dimeric Cr-peptide complex are identical to that of the biologically isolated fractions. Figure 1 Hypothetical models of low-molecular weight Cr peptide complexes. Results Genomic search for LMWCr Recent reports [ 18 , 21 , 24 ], using extracts from bovine liver, suggest that LMWCr is of peptide origin, having an approximate stoichiometric ratio consisting of E:D:G:C::4:2:2:2. In the absence of the exact sequence of this peptide, we generated all possible permutations matching the reported stoichiometry of LMWCr (10!/4!2!2!2! = 18,900) and performed an exact match search of the non-redundant ( nr ) protein database for their occurrence. Based on a simple model where all amino acids are equally possible and about 1.5 million sequences of average length 200 present in the nr database, the E-value for an independent exact match is about 0.55 [(0.05) 10 * 200 * 1.5 * 10 6 * 18,900]. We found perfect matches in the nr database corresponding to 3 unique permutations: EDGEECDCGE, DGEECDCGEE and CEGGCEEDDE. One match, CEGGCEEDDE, was found only in one sequence annotated as a putative protein kinase in rice ( Oryza sativa ). The peptide sequences, EDGEECDCGE and DGEECDCGEE, both matched the same region of the disintegrin domain of the human and mouse versions of ADAM 19 (A Disintegrin and Metalloproteinase domain 19; GeneID: 8728) [ 27 ]. Since the bovine form of ADAM 19 has yet to be sequenced, we were unable to determine if this sequence is also present in bos taurus . This acid-rich sequence region (Table 1 ) is present at the beginning of the disintegrin domain of ADAM19 [ 27 ]. We note that the DNA coding location for this subsequence is at the very end of an exon, giving rise to the speculation that the decameric peptide could possibly be an alternative splicing product. Arguments against this sequence giving rise to LMWCr and the complex acting intracellulary are that ADAM 19 is a membrane protein with extracellular domains. This argument is not needed if Cr might act extracellularly; the conserved nature of the motif leads one to suspect that it plays an important role, but it is at best a highly tentative candidate for being LMWCr. From these results, LMWCr appears unlikely to be a contiguous peptide sequence, unless an as-yet undiscovered peptide is found. Table 1 Multiple sequence alignment showing conservation of the EDGEECDCGE motif in all known mammalian forms of ADAM19 and a homologous putative ADAM in fission yeast (top). Multiple sequence alignment showing proposed Cr(III) binding sequence, NKDDNEECGD, conserved in the insulin receptor (INSR) across species, but not in the insulin-like growth factor receptor (IG1R) (bottom). SwissProt Protein Species Sequence Q9H013 ADAM19 Human GNGYL EDGEECDCGEEEE CNNP O35674 ADAM19 Mouse GNGYL EDGEECDCGEEEE CNNP ADAM19 Rat GNGYL EDGEECDCGEEDE CKNP O13766 Homolog S. Pombe GNGIV EDGEECDCGEDCE NNPC P15208 INSR Mouse NKDDN EECGD VCPGT P15127 INSR Rat NKDDN EECGD VCPGT P06213 INSR Human NKDDN EECGD ICPGT Q9PVZ4 Homolog Xenopus NRDNK EECGD VCPGT Q60751 IG1R Mouse NKPPKECGDLCPGTL P24062 IG1R Rat NKPPKECGDLCPGTL P08069 IG1R Human NKPPKECGDLCPGTL O73798 Homolog Xenopus NKPPKECVDLCPGA. Noting that the reported stoichiometry consists of even numbers of amino acids, we further considered the possibility that LMWCr might be a disulfide-linked dimer, with each monomeric unit having the composition, E:D:G:C::2:1:1:1. Such a hypothesis is consistent with the limited resolution of the experimental data used to derive the stoichiometry [ 24 ] and yields 60 sequence permutations. The expected number of random matches in the nr database is much higher, ~10 4 . Restricting this search to the human proteome gave rise to 439 (expected: ~10 2 ) matches, with no reason to prefer one over the other. Insulin signaling pathway mapping Considering that LMWCr might be a subsequence of a protein related to "insulin" or known to be involved in the insulin signalling pathway, we compiled a set of 96 such sequences. This set comprised two components: 1) proteins playing a role in the insulin signaling pathway (23 protein sequences were selected) derived from pathway charts, and 2) the set of all protein sequences derived from SwissProt for the search, "insulin + human" (78 entries). The two components were compared for redundancy and the duplicates removed (5 instances). A cross comparison of the resulting 439 entries using the BLAST query method versus the set of 96 pathway and insulin-related proteins results in a unique match for one of the 60 pentapeptides, EECGD, within the insulin receptor (INSR), residues 180–184 (expected matches: ~10 -1 ). Comparison of the pentapeptide set to a more detailed insulin signaling pathway construct [ 28 ] yielded the same result. This sub-sequence lies in the extracellular α-subunit of INSR in an acid-rich region (-NKDDNEECGD-) towards the end of the L1 domain, and at the start of the "cysteine-rich region". A BLAST query [ 29 ] for all INSR homologs in the nr database shows that this sub-sequence and acidic region is conserved in mouse and rat. Given the location of this acidic sequence within a molecule central to glucose homeostasis, the correspondence with experimentally measured stoichiometry and conservation across multiple species, we speculate that this sequence, or a fragment from this acid rich region may give rise to Cr-peptide fractions isolated from tissue. This suggestion implies that such fractions may not be homogeneous, discrete Cr(III) complexes, i.e. proteolysis may lead to a group of similar peptides that differ by one or more amino acid residues on either side of the Cr binding site. This interpretation differs significantly from reports on the isolation of LMWCr, which attempt to avoid a proteolytic product. A crystal or solution structure of this region of the insulin receptor has not been determined. However, the crystal structure of a homologous molecule, the insulin-like growth factor receptor (IGR1, GeneID: 124240) has been published [ 30 , 31 ]. Sequence alignment shows that this molecule exhibits a conserved difference in this region, having the subsequence -KECGD- in the same region instead of -EECGD- as found in INSR. The decameric "acidic region" found in INSR is not present in this growth factor receptor – cf. -NKPP KECGD LCPGTL-. The cysteine in -KECGD- forms a disulfide bond with another cysteine residue 28 positions away in the crystal structure. However, we do not know if this is due to the crystallization conditions or representative of the natural form. Further, the insulin receptor possesses 3 additional cysteine residues compared to the insulin-like growth factor receptor, thus the pattern of pairing of cysteine residues to form disulfide bonds may be different in the two receptor types. The observed difference may be great enough to preclude Cr(III) acting on the insulin-like growth factor receptor. Chromium-peptide complex synthesis The identified sequence in hand, we synthesized the pentapeptide (AcEECGD-CONH 2 ) and generated the disulfide-linked dimer via air oxidation. This peptide was subjected to conditions for the reconstitution of apo-LMWCr to generate a holo-peptide complex. Incubation of the peptide with fresh solutions of CrCl 3 results in a clear gray-green solutions of a Cr-peptide complex with visible electronic spectra (Figure 2 ) consistent with those reported for LMWCr [ 24 ]. Further, EPR experiments with Cr-peptide complexes generated from (AcEECGD-CONH 2 ) 2 and (AcNEECGD-CONH 2 ) 2 indicate that Cr exists in trinuclear arrays in these complexes. A preliminary spectrum is shown in Figure 2 consistent with that of isolated LMWCr fractions [ 32 ], further characterization is necessary to identify the exact nature of the complexes in these experiments. Figure 2 Schematic of proposed Cr-peptide complex. The EPR spectrum (top) is comparable to that of isolated LMWCr with g ~2.0; the spectrum also includes additional hyperfine coupling. Visible absorption spectra of complex formation from the reconstitution of apo-peptide with fresh solutions of chromium chloride (bottom). These data are qualitative and may comprise more than one discreet species. Discussion Barring the discovery of a novel, unsequenced or unidentified protein or peptide, these data point to the possible sequence of LMWCr fractions and may point to new strategies in therapeutic design. In addition, the question of sequence specificity in Cr(III)-peptide complexes must be fully addressed, along with thermodynamic and kinetic aspects of Cr(III) binding and transfer. Models of non-toxicological action of Cr(III) in biological systems may broadly fall into three categories: structural, redox, and iron homeostasis. The earliest models [ 12 ], and those advanced by Vincent [ 18 ], fall into a structural category and focus on the interactions of Cr(III) with peptides and proteins to affect insulin signaling and glucose metabolism, either directly or indirectly. There is an important redox model recently advanced [ 33 ] that suggests higher oxidation states of Cr interact with tyrosine phosphatases to inhibit the down-regulation of the insulin receptor. Finally, the chemical similarity of the Cr(III) and Fe(III) cations, and various in vitro studies suggest that Cr(III) replacement in the physiological iron transport and storage apparatus may lead to some small beneficial outcome for certain diseases [ 10 ]. The biological relevance of these models, and of in vitro experiments (including our own) may be finally ascertained only after the fact. Although unexpected, the results in this report and a critical review of other literature [ 9 , 11 - 18 ], suggest that an extracellular model for Cr(III) biochemistry with respect to insulin signaling may be plausible (see Supporting Information). Such a structural model would include the known aspects of INSR cycling and insulin degradation [ 34 ], and include the proposed interactions between Cr(III) and the INSR at the acidic site identified by our genomic search. This model is reminiscent of Mertz and co-workers' original proposal [ 12 ] of a ternary interaction between Cr(III), insulin and insulin receptor. It is substantially different from intracellular mechanisms for LMWCr action [ 16 , 18 ], redox mechanisms [ 33 ], and the iron homeostasis model [ 10 ]. In addition, the cycling of the insulin receptor and insulin degradation [ 34 ] may satisfy the problems of cellular distribution of Cr(III) and production of LMWCr via proteolysis. Experimentally observed insulin potentiating activity of Cr(III) may result from binding to the alpha subunit or bridging interaction between the two α subunits of an intact INSR molecule. This model is a parsimonious alternative to current proposals of Cr action in the insulin signaling pathway. However, this model points directly back to significant kinetic and a thermodynamic questions about Cr(III) in biological systems. For instance, what is the physical form of Cr in the bloodstream? How is Cr(III) transported and exchanged between ligands in the serum? Is transport specific? What structure/activity relationship exists in Cr(III) complexes to allow their transport across biological membranes? Thermodynamically, a hydrolyzed, multinuclear Cr cluster should predominate at neutral pH, but transport by transferrin would presumably be in the mononuclear Fe binding sites. Alternatively, Cr(III) clusters may be transported non-specifically in serum by proteins, possibly including transferrin and serum albumin. At this point, there exist significant gaps in understanding the possible biochemistry of Cr(III) and what molecular processes it may affect. The proposed extracellular model of Cr(III) action in this report is upstream of IRS1, a therapeutic target of White and others [ 7 , 8 ], and may lend itself to small-molecule therapeutic strategies for diabetes and other metabolic conditions [ 1 ]. We hope this model may pave the way for innovative experiments, better models of Cr(III) biochemistry and excretion, and further understanding of signaling events in complex biochemical pathways. Conclusions A bioinformatic search for an acidic decameric sequence matching reported stoichiometries of LMWCr amino acid composition indicates that the peptide may not be a contiguous sequence. An expanded search localized a pentameric sequence in the insulin receptor and suggests a possible identity for the Cr(III)-containing peptide fractions derived from liver. Disulfide linked penta- and hexameric peptides based on the identified sequence bind Cr(III) in a similar fashion to LMWCr fractions reported in the literature. Methods Search for LMWCr The nr database was downloaded and Perl scripts used to search for exact matches corresponding to all unique permutations of EEEEGGCCDD. In a separate search, a set of 78 unique protein sequences from Swiss-Prot were obtained as a result of using the query "insulin human." This set was searched for exact matches corresponding to all unique permutations of EEGCD. In addition, the human proteome was also downloaded and searched for matches to the same set of pentameric peptides. N-Acetylglutamylglutamylcysteinylglycylaspartyl carboxamide (AcEECGD-CONH 2 ) The peptide, AcEECGD-CONH 2 , was synthesized by continuous-flow automated solid-phase synthesis on a Perseptive Biosystems Pioneer Peptide Synthesis System. Peptide synthesis was performed by using standard Fmoc-protection strategies with TBTU/DIEA activation strategy. Typically, a 0.5 mmol scale, using Rink amide resin (loading ~0.65 mmol/g) and four times excess of the other reagents (TBTU, protected amino acid) were used. After the solid-phase synthesis was complete, acetylation at the amino terminus was carried out for 2 hours (50:50:1:: acetic anhydride:dimethylformamide:pyridine), and the resulting peptide cleaved from the resin. The peptide was cleaved from the solid phase resin by adding a slurry of 94% trifluoroacetic acid, 2.5% of water, 2.5% of ethanedithiol, and 1% of triisopropylsilane to the reaction vessel and shaking it for 5 h. The trifluoroacetic acid, containing the peptide, was filtered off by vacuum, and the resin was washed 2–3 times with trifluoroacetic acid. The filtrate was evaporated under nitrogen gas until the volume was reduced to 15 mL. 30 mL of ice-cold diethyl ether was added to the filtrate, causing the peptide to precipitate, and the mixture centrifuged to form a pellet of the peptide. The diethyl ether was decanted and the peptide pellet was washed with diethyl ether three times. Finally, the peptide was dissolved in 20 mL water containing 0.1% trifluoroacetic acid and extracted with diethyl ether three times. The aqueous portion was collected and freeze-dried to give the synthetic peptide. AcEECGD-CONH 2 , has a retention time of 5.3 minutes on a 250 mm × 4.5 mm C-18 reversed phase HPLC column using a gradient of 5 to 20% acetonitrile in 0.1% trifluoroacetic acid/water mobile phase running at 1 mL per minute and detected at a wavelength of 220 nm. N-Acetylglutamylglutamylcystinylglycylaspartylcarboxamide dimer ((AcEECGD-CONH 2 ) 2 ) The synthetic peptide, AcEECGD-CONH 2 , was dissolved to 10 mg/mL in a 0.1 M solution of ammonium carbonate at pH 7 and allowed to oxidize under ambient air for 48 hours. The product was isolated by lyophilization and analyzed by HPLC. The disulfide peptide dimer, (AcEECGD-CONH 2 ) 2 , has a retention time of 6.0 minutes using the above conditions. The product, (AcEECGD-CONH 2 ) 2 , has a calculated mass of 1183.35141 and exhibits a mass of 1183.3477 Daltons when analyzed by electrospray mass spectrometry. Chromium N-acetylglutamylglutamylcystinylglycylaspartylcarboxamide (Cr 3 O(AcEECGD-CONH 2 ) 2 ) Fifteen milligrams of (AcEECGD-CONH 2 ) 2 was weighed and dissolved in 30 mL of water. A portion of this solution (5 mL) was taken up in a 25 mL tube, and 4 equivalents of chromium(III) chloride were added as a solid or in an aqueous solution. The reaction of the two components took several minutes and was monitored by ultraviolet-visible spectrophotometry. The chromium peptide complex, Cr 3 O(AcEECGD-CONH 2 ) 2 , exhibits characteristic spectral absorbance features at 432 nm and 615 nm. The features are consistent with chromium bound to oxygen atom donors and similar to the reported spectrum of LMWCr [ 24 , 32 ]. EPR spectra were collected using the following parameters: microwave frequency, 9.632 GHz; microwave power incident to the cavity, 2 mW; temperature, 10 K (LHe cryostat). Samples were prepared by incubating a solution of peptide with chromium chloride at a final concentration of 1 mM in metal with excess peptide. The complete characterization (EPR, MS, etc.) of this and analogous Cr-peptide complexes will be reported elsewhere. Authors' contributions DD constructed informatics scripts and searches and made sequence alignments. VM and KA constructed pathway maps and conducted informatics searches. SC assisted in peptide synthesis and conducted HPLC analysis. VM and BB collected ESR spectra. JDVH conceived of the study, synthesized peptides and Cr(III) complexes and drafted the manuscript, tables and graphics. All authors read and approved the final manuscript. Supplementary Material Additional File 1 Supporting figures (3) include HPLC and MS data for AcEECGD-CONH 2 and proposed Cr(III) model at INSR. Supporting tables (2) include FASTA sequences for insulin signaling map (20 proteins) and pentameric peptides found in genomic search (439 entries) Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC539358.xml
535539	The Israeli strain IS-98-ST1 of West Nile virus as viral model for West Nile encephalitis in the Old World	West Nile virus (WNV) recently became a major public health concern in North America, the Middle East, and Europe. In contrast with the investigations of the North-American isolates, the neurovirulence properties of Middle-Eastern strains of WNV have not been extensively characterized. Israeli WNV strain IS-98-ST1 that has been isolated from a white stork in 1998, was found to be highly neuroinvasive in adult C57BL/6 mice. Strain IS-98-ST1 infects primary neuronal cells from mouse cortex, causing neuronal death. These results demonstrate that Israeli strain IS-98-ST1 provides a suitable viral model for WNV-induced disease associated with recent WNV outbreaks in the Old World.	West Nile virus (WNV) is a single-stranded RNA flavivirus (family Flaviviridae , genus flavivirus) with a worldwide distribution ranging Africa, Europe, the Middle East, and Asia. WNV was first recognized in the Western Hemisphere in 1999. The emergence of WNV has been associated with a dramatic increase in severity of disease in humans and other species[ 1 , 2 ]. Recent WNV epidemics which include meningitis, encephalitis and poliomyelitis-like syndrome in humans have been reported in Europe, the Middle-East and in North America. During the summers of 2002 and 2003, more of 13,000 human cases and 500 deaths were reported from the United States, drawing the attention of WNV illness as an important public health concern. Comparison of WNV strains identified two major genetic subtypes: the lineage II (enzootic strains from tropical Africa and Madagascar island) and the lineage I (tropical african strains) that caused the outbreaks of WNV infection in North Africa, Europe, Israel, and in the United States. Nucleotide sequencing revealed that American strains of WNV isolated between 1999 and 2000 are nearly identical to Israeli strains of WNV isolated in 1998 and 2000 [ 3 , 4 ]. This close relationship could be explained by the fact that an Israeli WNV strain was introduced in New York City in 1999 [ 4 ]. The murine model of WNV-associated encephalitis has been widely used to address the viral pathogenesis[ 5 ]. Strains of WNV isolated in the United States were found to be highly neuroinvasive in adult mice following intraperitoneal (i.p.) inoculation[ 6 ]. In contrast of the investigations of the North-American WNV strains, the virulence phenotype of Israeli strains of WNV has not been extensively characterized. The WNV strain IS-98-ST1 has been isolated from cerebellum of a white stork during an outbreak in Israel in 1998[ 7 ]; its phenotypic characterization was performed after 3 passages in the mosquito cell line Aedes pseudoscutellaris AP61[ 8 ] and its complete genomic sequence determined (GenBank accession number AF481864). Virus titration was performed on AP61 cells by focus immunodetection assay as previously described [ 9 ]. Infectivity titers were expressed as focus forming units (FFU). In this study, we demonstrated that IS-98-ST1 has a high neuroinvasive potential in adult C57Bl/6 mice, and that the virus is capable to replicate in primary neuronal cultures from mouse brain cortex. Mouse experiments were performed according to the European Convention 2001–486. After anesthesia, six-week-old female C57BL/6 mice (Harlan, France) were inoculated with 1,000 FFU of WNV via different routes (15 animals per group): intraperitoneal (i.p.), intradermal (i.d.), intracerebral (i.c.), and intranasal (i.n.). At Days 5 and 7 of infection, three animals per group were euthanasied; brain and spinal cord were rapidly removed, processed for viral titration or sectioned on cryostat (Jung Frigocut; 14 μm thick sections). Sections were fixed with 3.7% formaldehyde or acetone for 30 min and processed for indirect immunofluorescence with mouse polyclonal anti-WNV antibodies[ 8 ]. Some sections were also processed for Glial Fibrillary Acidic Protein (GFAP) using a rabbit polyclonal antibody (Promega). Sections were further washed, mounted and observed with a fluorescence microscope (DMRB Leica). When infected i.c., mice died at day 7.3 ± 1 post-infection (p.i.) ; 100% mortality was also reached after i.p., i.n., or i.d. inoculation but with delayed kinetics (day 9.5 ± 0.5, 10.7 ± 0.7 and 10.5 ± 0.5 p.i. respectively). In all cases, WNV-infected mice exhibited characteristic disease progression with hind limb paralysis, cachexia and tremors. By day 7 p.i., WNV was found in brain tissue in all mice, reaching virus titers from 3.10 5 (i.d. route) to 3.10 8 FFU/g (i.c. route). To investigate WNV location within the CNS, cryostat brain sections from three WNV-infected mice were assessed for the presence of viral antigens by immunofluorescence at day 7 p.i. When inoculated i.c, virus was found widespread in most of the brain structures (whereas no signal was seen in mock-infected controls), including cortex (Fig. 1A ), pyramidal neurons of the hippocampus (Fig. 1B ), spinal cord and olfactory bulb. In contrast, a lower level of infection was observed after i.p., i.d. or i.n. inoculation (Fig. 1E ), showing regional variations according to the route of inoculation (Fig. 1C and 1D ). In all sections, WNV-infected cells were negative for GFAP (Fig 1F ). This suggests that neurons are the principle targets of infection in the CNS. Figure 1 A to F : WNV antigens in different regions of the mouse CNS. Mice were inoculated with 10 3 FFU of IS-98-ST1 WNV upon different routes (i.c., i.p., i.n., i.d.); at Day 7 of infection, mice were euthanazied, brains were cut in 14 μm thick cryostat sections, and processed for immunofluorescence using anti-WNV serum (obtained from i.p.-inoculated resistant mice) as primary antibody. A : hippocampus (pyramidal layer), i.c. inoculation. B : frontal cortex, i.c. inoculation. C : spinal cord, i.p. inoculation. D : olfactory bulb, i.n. inoculation. Magnification: × 350. E : Average levels of infection of the different brain structures was estimated on 10 different sections for each of the 3 animals per group (I.C.: intracerebral, I.P.: intraperitoneal, I.D.: intradermal; I.N.: intranasal) according to the scale: +++: more than 10 positive cells per microscopic field; ++: between 3 and 9 positive cells; +: 1 or 2 positive cells; -: no positive cell. F : Immunodetection of WNV antigens (green) and Glial Fibrillary Acidic Protein (red) in cryostat section of WNV-infected mouse brain, day 7 of infection, i.c Magnification: × 700. G, H : WNV infection in primary neural cultures from C57BL/6 mouse brain cortex. Primary cultures were performed as described in text and infected with IS-98-ST1 WNV. G : Detection of WNV antigens (using anti-WNV mouse immune serum and a FITC-conjugated secondary antibody, green staining) and neuronal specific enolase (using a rabbit polyclonal antiserum and an anti-rabbit polyclonal antibody made in goat conjugated with Texas Red, red staining) by immunofluorescence at 24 h p.i. (m.o.i. 12.5). Magnification: × 700. H : Kinetics of infection and variation of cell number at various times post-infection for different m.o.i; three cultures for each m.o.i. were fixed and processed for WNV antigen detection by immunofluorescence, whereas cell nuclei were visualized with DAPI. Cell nuclei of adherent cells were counted in 8 different different fields for the three cultures (histogram) whereas the percentage of infected cell was estimated by counting WNV antigen positive cells and cell nuclei; the percentage of infected cells is indicated as values (%) in white squares. For ex-vivo experiments, primary neuronal cultures were prepared from the brain cortex of C57/BL6 mouse embryos (day E15) (Harlan, France)[ 10 ]. Briefly, after rapid removal of the embryos and dissection of brain cortex, mechanical dissociation and centrifugation were performed; the cells were seeded on slides and grown in NeuroBasal/B27 medium (Invitrogen Corporation) and, around 10 days after plating, were infected with WNV at different multiplicities of infection (m.o.i.). Cell cultures were constituted by more that 90% neurons, as assessed by immunocytochemistry. At different times post-infection, cell culture supernatants were processed for viral titration; cells were fixed and processed for immunofluorescence detection for viral antigens (see above) or neural cell typing, using either an anti-neuron specific enolase (NSE) (Zymed) or an anti-GFAP (Promega). After 24 h of infection at a m.o.i of 25, ~50% of cells were infected (Fig. 1H ). By 40 h p.i., 90% of cells became infected and > 10 7 FFU of WNV per ml was detected in the culture supernatant. Time course studies showed that IS-98-ST1 infection induced cell death through neuronal necrosis within 48 h of infection, and ~90% of cells had detached by 96 h (Fig. 1H ). Whatever the time of infection, only neuronal cells were permissive for IS-98-ST1 as judged by double immunofluorescence staining for WNV antigens and NSE (Fig. 1G ). GFAP positive cells, i.e. astrocytes, that constitute less than 10% of cells appeared to be relatively resistant ot WNV infection. To confirm this, astrocyte-enriched primary cultures from the brain cortex of mouse embryos were infected with IS-98-ST1 at a m.o.i of 50. By 48 h p.i., only 5% of GFAP immunoreactive cells expressed viral antigens (data not shown). Although our study was limited in its scope, the results indicate that WNV strain IS-98-ST1 is suitable as viral model for West Nile encephalitis in the Old World. The Israeli strain IS-98-ST1 that caused the epizootic in Israel in 1998, was found to be highly neuroinvasive in mice following peripheral inoculation. Consistent with this observation, we reported that IS-98-ST1 has an i.p. LD50 value as low as 10 FFU[ 8 ]. IS-98-ST1 infection has allowed us to determine the role of the type-I interferon (IFN) response in controlling WNV infection and that IFN-inducible OligoAdenylate Synthetase molecules may play an important role in the innate defense mechanism against WNV[ 8 , 11 ]. High viral titers could be recovered in mouse brains whatever the route of inoculation (i.c., i.p., i.d., i.n.). Viral antigens were detected in most brain structures at day 7 of infection, consistent with the notion that IS-98-ST1 is able to reach the CNS and then replicate in the brain. Infected C57Bl/6 mice showed neurological symptoms and lethality, confirming the high neurovirulent characteristics of IS-98-ST1, that were described in another susceptible mouse model of WNV (North-American strain) infection [ 12 ]. These features may be linked to the predominance of neurological symptoms that have been observed in hospitalized patients during Israeli outbreaks [ 13 ] or during natural infections of horses [ 14 ]. Our data are compatible with a previous report[ 15 ] indicating that WNV replicates locally in draining lymph nodes in mice inoculated subcutaneously, then in the spleen and in multiple sites in the CNS, although the sites of extraneural viral infection and the possible cells that could be involved in such a passage remain elusive. The dissemination of foci of infection within the brain that is observed in our study is compatible with virus passage through the blood-brain barrier. However, the fact that infected neural cells are detected in the olfactory bulb after intra-nasal inoculation suggests that an intraneural transport of WNV cannot be ruled out. Such neuroinvasive properties have also been reported for WNV variants from North America in experimental infection in rodents [ 16 ] and avian species as well as in natural infections in horses or birds[ 5 , 17 ]. Although some of these studies support the infection of neural cells by WNV within the CNS, none used double immunocytochemistry for WNV antigen and cell typing. Our study confirms the neurotropism of WNV and the huge preferential infection of neurons in vivo . Because neurons are believed to be main target neural cells of WNV, we developed an ex-vivo model of infection, by culturing primary neural cells from the brain cortex of susceptible mice. More than 90% of the neurons are found to be infected by IS-98-ST1 and infected neurons undergo necrosis. In contrast, astrocytes were mainly resistant to WNV infection. This is consistent with in vivo data showing a massive infection of brain structures such as brain stem, hippocampus and cortex of WNV-infected animals [ 12 ] and human patients[ 5 ]. The high neuropathogenicity of IS-98-ST1 isolated from a stork in Israel in 1998, as well as WNV strains present in North America does contrast with the low pathogenicity of most ancestral strains of WNV[ 18 , 19 ]. In conclusion, the Israeli strain IS-98-ST1 of WNV provides a relevant model for assessing the identification of viral factors that may responsible for West Nile pathogenesis. Authors' contribution ML carried out ex-vivo studies, M-PF and TM participated in in vivo experiments, VD and J-LG revised critically the article, PD and P-EC have written, drafted the article, and participated to in vivo and ex-vivo experiments. Competing Interests The authors declare that they have no competing interests.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535539.xml
549196	Use of a recombinant Salmonella enterica serovar Typhimurium strain expressing C-Raf for protection against C-Raf induced lung adenoma in mice	Background Serine-threonine kinases of the Raf family (A-Raf, B-Raf, C-Raf) are central players in cellular signal transduction, and thus often causally involved in the development of cancer when mutated or over-expressed. Therefore these proteins are potential targets for immunotherapy and a possible basis for vaccine development against tumors. In this study we analyzed the functionality of a new live C-Raf vaccine based on an attenuated Salmonella enterica serovar Typhimurium aroA strain in two Raf dependent lung tumor mouse models. Methods The antigen C-Raf has been fused to the C-terminal secretion signal of Escherichia coli α-hemolysin and expressed in secreted form by an attenuated aroA Salmonella enterica serovar Typhimurium strain via the α-hemolysin secretion pathway. The effect of the immunization with this recombinant C-Raf strain on wild-type C57BL/6 or lung tumor bearing transgenic BxB mice was analyzed using western blot and FACS analysis as well as specific tumor growth assays. Results C-Raf antigen was successfully expressed in secreted form by an attenuated Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. Immunization of wild-type C57BL/6 or tumor bearing mice provoked specific C-Raf antibody and T-cell responses. Most importantly, the vaccine strain significantly reduced tumor growth in two transgenic mouse models of Raf oncogene-induced lung adenomas. Conclusions The combination of the C-Raf antigen, hemolysin secretion system and Salmonella enterica serovar Typhimurium could form the basis for a new generation of live bacterial vaccines for the treatment of Raf dependent human malignancies.	Background The Raf proteins (A-Raf, B-Raf, C-Raf) are located upstream of MEK and downstream of Ras and represent an essential part of the mitogenic cascade [ 1 - 3 ]. Interestingly, Raf kinases are not only central players in cellular signal transduction, but are often causally involved in the development of cancer. Recently, B-Raf was found to be mutated in a broad range of malignancies including melanoma (more than 65%), and colon cancer [ 4 ]. In addition, overexpression of C-Raf was found in many tumors [ 5 , 6 ]. Therefore, these proteins are potential targets for immunotherapy as well as immunoprevention of tumors. Here we describe the development of a C-Raf vaccine on the basis of an attenuated Salmonella enterica serovar Typhimurium aroA strain. Such recombinant live vaccines have been shown to efficiently elicit both, humoral and cellular immune responses against a variety of heterologous antigens [ 7 , 8 ]. In order to achieve stable expression of C-Raf we used the E. coli α-hemolysin (HlyA) secretion system which is fully active in Salmonella [ 9 ]. This transport machinery is the prototype of type I secretion systems and consists of three different components, namely HlyB, HlyD and TolC. The HlyA carries at its C-terminus a secretion signal of about 50–60 amino acids in length (HlyA s ), which is recognized by the HlyB/HlyD/TolC-translocator, leading to direct secretion of the entire protein into the extracellular medium without the formation of periplasmic intermediates. In addition, fused to the C-terminus of heterologous proteins, the HlyA s leads to efficient secretion of such proteins by the recombinant bacteria. In our case, the whole C-Raf antigen fused to the C-terminal secretion signal of hemolysin was efficiently expressed and secreted by an attenuated Salmonella enterica serovar Typhimurium aroA strain. The effects of this recombinant vaccine were assessed in wild-type C57BL/6 or tumor bearing transgenic BxB mice. Methods Bacterial strains, plasmids, cell lines and mice The bacterial strains, plasmids, cell lines and mice used in this study are listed in Table 1 . Plasmid transformation in Salmonella enterica serovar Typhimurium SL7207 The plasmids pMOhly1 and pMOhly-Raf were first transformed in competent Salmonella enterica serovar Typhimurium LB5000 (Tab. 1 ), a restriction-negative and modification-proficient strain by a standard transformation protocol for E. coli [ 10 , 11 ]. Subsequently, the plasmids purified from Salmonella enterica serovar Typhimurium LB5000 were introduced into Salmonella enterica serovar Typhimurium SL7207 by electroporation using a Bio-Rad Gene Pulser (Hercules, CA, USA) at 2.5 kV, 25 microfarads (μF), and 200 Ohm in a 0.1 cm electroporation cuvette. Construction of the plasmid pMOhly-Raf Sense primer 5' Raf: 5'-ATGGAGCACATAC ATGCAT CTTGGAAG-3' and antisense primer 3'Raf: 5'-CAACTAGA ATGCAT GCAGCCTCGGGGA-3' were used to amplify by PCR a 1950 bp DNA fragment representing the entire craf gene from plasmid pUC13-c-raf-1 [ 12 ]. PCRs were performed in a Thermal Cycler 60 (Biometra, Göttingen, Germany) for 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 90 s. After purification with the GeneClean Kit (Bio101, La Jolla, Ca) and digestion with the NsiI restriction enzyme, the DNA fragment carrying the craf gene was inserted into the single NsiI site of the export vector pMOhly1 [ 13 ]. The resulting plasmid pMOhly-Raf was isolated from E. coli DH5α (Invitrogen), analyzed and transformed in S. typhimurium SL7207 (Tab. 1 ) by electroporation. Western blot analysis Salmonella enterica serovar Typhimurium strains harbouring the recombinant plasmids pMOhly1 or pMOhly-Raf were cultivated at 37°C in BHI medium with 100 μg/ml ampicillin. Cells at the exponential growth phase (OD 600 of 1) were centrifuged at 5000 × g at 4°C for 5 min. The supernatant proteins were precipitated with 10% (V/V) trichloroacetic acid (TCA) for 1 h on ice, collected by centrifugation and resuspended in SDS sample buffer. The supernatant proteins were separated on 10% gels by SDS-PAGE [ 14 ]. For immunodetection of Raf-HlyA proteins the rabbit polyclonal anti-Raf antibody SP-63 (diluted 1:1,000, Rapp Laboratory) and donkey anti-rabbit immunoglobulins linked to horseradish peroxidase (Amersham Pharmacia Biotech) diluted 1:1,000 as secondary antibodies were used. Blots were developed by enhanced chemiluminescence (ECL reagents; Amersham Biosciences, UK) and exposed on X-ray film (Kodak, XO-MAT-AR) for 1 min. Immunization of mice with Salmonella enterica serovar Typhimurium SL7207 strains Oral/intravenous prime boost protocol (p.o./i.v.) In order to achieve a broad immune response encompassing both, the mucosal and systemic immunity against C-Raf, we combined oral immunization (p.o.) with an intravenous (i.v.) boost. In these experiments, seven weeks old C57BL/6 or BxB23 mice were immunized, first p.o. three times with 5 × 10 9 bacteria/100 μl phosphate-bufferd saline (PBS) at 5-day intervals. At day 45 after the start of the vaccination, these mice were boosted intravenously (i.v.) with a single-dose of 5 × 10 5 bacteria/100 μl PBS. The BxB23 mice received a second i.v. boost of 5 × 10 5 bacteria at day 90 after the start of the vaccination. Induction of Raf-specific immune responses were analyzed at day 50 for C57BL/6 or at day 95 for BxB23 respectively. Intranasal Immunization (i.n.) Seven weeks old BxB23 mice were immunized i.n. four times with 1 × 10 7 bacteria/30 μl phosphate-buffered saline (PBS) at 14-day intervals. The vaccine was applied using a micropipette onto the nares of mice under anesthesia. Induction of Raf-specific immune responses were analyzed at day 70. I. n. Immunization of BXB11 mice with Salmonella enterica serovar Typhimurium SL7207/pMOhly-Raf Four months old BXB11 mice were immunized i.n. three times with 1 × 10 8 salmonellae in 10 μl PBS at 14-day intervals. The vaccine was applied using a micropipette into the nares of mice without anesthesia. Construction of a C-Raf overexpressing EL-4 cell line In order to create tools for analysis of C-Raf specific T cells we first constructed a C-Raf overexpressing EL-4 cell line (ELRaf). The EL4 tumor cell line is a murine thymoma cell line of the H-2 b haplotype (Tab. 1 ). 10 μg purified DNA of plasmid pDNA3craf was introduced into EL4 cells by electroporation with the Bio-Rad Gene Pulser (Hercules, CA) at 0.25 kV, 960 μFD, in a 0.4-cm electroporation cuvette. The transformed cells were grown RPMI 1640 medium (Invitrogen) supplemented with 5% (vol/vol) FCS (PAN Systems) and 600 μg/ml G418 (Sigma). C-Raf is highly expressed in ELRaf cells (data not shown). Flow cytometric detection of specific CD8 + T-cells Isolation of spleen cells Animals were sacrificed and a single cell suspension of splenocytes was prepared by passage of the spleen through a sieve into RP 10 medium [RPMI medium (Life Technologies) supplemented with glutamine (1%), 50 μM β-mercaptoethanol (ROTH, Wuerzburg), penicillin (10 U/ml, GIBGO), streptomycin (100 U/ml, GIBGO) and 10% fetal calf serum (PAN™, BIOTECH GmbH). The cell suspension was centrifuged and resuspended in 3 ml lysis buffer (5 mM Tris-HCl, 140 mM NH 4 Cl, pH 7.3) for the lysis of erythrocytes. After 2 minutes, 10 ml RP 10 medium was added to stop lysis. After centrifugation, cells were resuspended in 2 ml RP 10 medium and counted. Restimulation In 6 ml FACS tubes and a total volume of 100 μl RP 10 medium in the presence of 30 U/ml recombinant IL-2 and 10 μg/ml Brefeldin A at 37°C, 5% CO 2 , 1 × 10 6 spleen cells were incubated for 5 hours with 5 × 10 5 C-Raf overexpressing EL-4 cells (EL-Raf) or 5 × 10 5 EL-4 cells or with the addition of 10 ng/ml of phorbol myristyl acetate (PMA; Sigma) and 500 ng/ml of ionomycin (Sigma) or with medium alone. Staining After restimulation, cells were washed with PBS-0.1% bovine serum albumin (BSA; P-B buffer) and incubated with 1 μl of anti-CD8-CyChrome (Pharmingen Nr. 01082A) in a volume of 100 μl PBS-0.1% BSA for 20 min on ice. Subsequently, cells were washed and fixed for 20 min at room temperature with PBS-2% paraformaldehyde (Sigma). After washing with PBS-0.1% (BSA), cells were permeabilized with PBS-0.1% BSA-0.5% saponin (Sigma, P-B-S) buffer. After incubation for 5 min at room temperature, cells were washed with P-B-S buffer and incubated in a volume of 100 μl at room temperature with polyclonal rat immunoglobulin G (IgG) antibodies (JacksonImmunoResearch) to block nonspecific binding and 1 μl anti-IFN-γ-FITC (AN18.17.24; XMG1.2 Rat IgG1; Pharmingen) for 20 min. After incubation, cells were washed twice with P-B-S buffer and another time with P-B buffer. Cells were resuspended in 400 μl PBS-PFA 0,5% and kept at 4°C until analysis. Analysis Cells were analyzed by flow cytometry in a FACS Calibur flow cytometer (Becton Dickinson) using CellQuest 3.0 software (Becton Dickinson). Lymphocytes were chosen according to their size and granularity in a forward/side – scatter diagram. Numbers are expressed as percent IFN-γ positive CD8 + cells. Statistical analysis The statistical significance of differential findings between experimental groups was determined by Student's t test. Findings were regarded as significant, if P values were <0.05. Survival curves were compared using a log rank test. Results Creation of a C-Raf vaccine on the basis of the attenuated Salmonella enterica serovar Typhimurium strain SL7207 The construction of the attenuated Salmonella enterica serovar Typhimurium aroA strain SL7207 secreting the C-Raf antigen was achieved by cloning the human craf cDNA from pUC13-c-raf-1 [ 12 ] into the vector plasmid pMOhly1 [ 13 ] as described in materials and methods. The resulting plasmid pMOhly-Raf carried the craf-hlyAs fused gene and the functional hlyB and hlyD genes required for its secretion (Fig. 1 ). The S. typhimurium SL7207/pMOhly-Raf strain efficiently expressed and secreted the hybrid C-Raf protein, as shown by immunoblotting with polyclonal antibodies raised against C-Raf (Fig. 2 ). The amount of secreted C-Raf was 2–3 μg protein/ml supernatant under the experimental conditions. Raf-specific responses of mice after immunization with recombinant SL7207/pMOhly-Raf The efficacy of the recombinant bacterial strain to induce a Raf-specific immune response was analyzed using wild-type C57BL/6 and transgenic BxB23 mice. Groups of five C57BL/6 and BxB23 mice at the age of 7 weeks were immunized p.o./i.v or i.n. with recombinant Salmonella enterica serovar Typhimurium SL7207 secreting C-Raf antigen (SL7207/pMOhly-Raf) and with Salmonella enterica serovar Typhimurium SL7207 as control in order to test the induction of Raf-specific immune responses. The data showed that 20% of the sera of BxB23 mice immunized with SL7207/pMOhly-Raf contained Raf-specific antibodies (Fig. 3 ; supplementary Fig. 1 [see Additional file 1 ]). In contrast, no Raf-specific IgG response was detectable in the sera of BxB23 mice immunized with SL7207 alone (data not shown). Similar data were obtained after immunization of C57BL/6 mice with SL7207/pMOhly-Raf and SL7207 (data not shown). To assess the induction of Raf-specific T-cell responses, mice were sacrificed and Raf specific T-cell responses were assessed using intracellular IFN-γ staining followed by FACS analysis. For this purpose, T-cells were restimulated with C-Raf overexpressing EL-4 cells. Using this technique, we could detect Raf specific CD8 + T-cell response in C57BL/6 animals immunized p.o./i.v. with SL7207/pMOhly-Raf only but not in mice immunized i.n. or with the control strain SL7207 (Fig. 4 ). In immunized BxB23 mice, but not naïve BxB23 mice, a high background and variability of CD8 + IFN-γ positive cells even with non-specific stimulation was observed in two independent experiments. Therefore it was not possible to assess specific T-cell responses in this setting. Interestingly, the level of T-cells which responded to polyclonal stimulation by PMA / ionomycin was also reduced about 4 fold in comparison to C57BL/6 mice (data not shown). Salmonella enterica serovar Typhimurium SL7207/pMOhly-Raf strain induced partial protection against lung cancer in transgenic mouse models of Raf oncogene-induced lung adenomas To test the protective capacity of the immune responses induced by the recombinant Salmonella strains, groups of 6 to 10 heterozygous BxB23 mice at the age of 7 weeks were immunized p.o./i.v. or i.n. with recombinant Salmonella enterica serovar Typhimurium strain (SL7207/pMOhly-Raf) or with SL7207 as control. BxB23 mice normally show an induction of lung adenomas with short latency and at 100% incidence [ 15 , 16 ]. The development of lung adenomas in the vaccinated BxB23 mice was assessed for 13 months. Our analysis revealed a significantly delayed tumor growth (reduction of lung weight) in mice immunized i.n. or p.o./i.v. with SL7207/pMOhly-Raf compared to control mice (Fig. 5 ). In order to confirm these data we repeated the protection experiments using BxB11, another craf transgenic strain, which develop lung adenomas after a shorter latency period compared to BxB23 mice [ 15 ]. In this case, 66% of the mice immunized with SL7207/pMOhly-Raf survived at least three months longer than the control mice (Fig. 6 ). These results suggest that the immunization with the vaccine strain SL7207/pMOhly-Raf can achieve a partial protection against tumor growth in the BxB mouse model. Discussion The major problems for vaccine development against cancer are the heterogeneity of the tumor cells and the fact that all tumor antigens are self-antigens. Therefore specific T-cells might be anergic or tolerant [ 17 , 18 ]. However, despite these problems, several cancer vaccines have already reached clinical trials [ 19 ]. The success of these vaccines seems to be dependent on the target antigen and on the tumor type. Here we describe a new strategy for achieving an anti-tumor immune response with a C-Raf vaccine on the basis of an attenuated Salmonella enterica serovar Typhimurium aroA strain as a "live vaccine". In general, recombinant aroA salmonellae are efficient live bacterial vectors that stimulate strong mucosal immunity, humoral and cell-mediated responses with a great potential as live vaccine carriers in both humans and animals [ 7 , 20 ]. Advantages of live attenuated aroA Salmonella vaccines include their safety and easy administration [ 21 , 22 ]. In addition aroA Salmonella enterica serovar Typhimurium strains were already successfully used as carriers for DNA vaccines against cancer in mice [ 23 ]. In this study the C-Raf antigen was delivered in secreted form by attenuated Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. This system allows an efficient antigen secretion and presentation, which are necessary for an optimal immune response against the given heterologous antigen [ 9 ]. In fact, immunization of mice with the Salmonella enterica serovar Typhimurium aroA strain SL7207 secreting C-Raf resulted in the induction of a humoral immune response, manifested by the presence of Raf-specific antibodies in some of the vaccinated mice. In addition, C57BL/6 mice immunized with Salmonella enterica serovar Typhimurium SL7207/pMOhly-Raf developed a Raf-specific CD8 + T-cell response. The C-Raf vaccine thus is able to break the peripheral tolerance of the immune system towards C-Raf and to induce a specific immune response. Although the transgenic model is closer to the human setting in comparison to challenge models with tumor cell lines, we can not formally exclude that lung specific expression of the transgene in our BxB model is not sufficient for the induction of peripheral tolerance. Furthermore, side effects due to autoimmunity might occur in humans who have a more generalized expression pattern. However, the lung tissue does not belong to immunoprivileged sites and we have never observed any signs of lung pathology in immunized animals which strongly suggests that the data could, in principle, be translated into the human setting. We demonstrated also a partial tumor protection both in BxB23 and in BxB11 mice after immunization with recombinant Salmonella enterica serovar Typhimurium SL7207/pMOhly-Raf. However, we were not able to assess Raf specific CTL responses in these mice due to the high variability and background and therefore we cannot conclude whether protection is really due to cytotoxic T-cells or might be the result of other effects, e.g. an increased intratumoral level of IFN-γ produced by Raf specific CD4 + T-cells. Interestingly, the observed variation occurred only in treated BxB23 mice, not in naïve BxB23 mice. Therefore, the observed effect might be due to a spontaneous induction of an immune response mediated by tumor infiltrating bacteria. It is interesting to note that spontaneous Raf specific immune responses also occur in the human context, which we have recently demonstrated B-Raf V599E specific CTL and B-Raf /B-Raf V599E specific humoral responses in melanoma patients [ 24 , 25 ]. Most importantly, the lack of B-Raf V599E mutations in metastases of melanoma patients with strong B-Raf V599E CD8 response [ 24 ] supports the notion that CD8 + T-cells are effective in eliminating antigen positive tumor cells. The combined data thus provide proof of concept for the development of Raf-based anti-cancer vaccines. This is the first example demonstrating that the E. coli hemolysin secretion system is a versatile tool for the delivery of cancer antigens in Salmonella enterica serovar Typhimurium. Moreover, we have recently shown that this secretion system is also fully active in Salmonella enterica serovar Typhi Ty21a, the only Salmonella vaccine strain registered for human use [ 26 ]. Therefore, the combination of the hemolysin secretion system and S. typhi Ty21a could form the basis for a new generation of live bacterial vaccines against cancer. Conclusions Taken together we have demonstrated that the C-Raf antigen can be successfully expressed and secreted by the attenuated Salmonella enterica serovar Typhimurium aroA SL7207 strain via the E. coli hemolysin secretion system. In addition, the immunization of wild-type C57BL/6 or tumor bearing transgenic mice with this C-Raf secreting Salmonella strain provoked specific C-Raf antibody and T cell responses. Most importantly, the vaccine strain induced partial protection against lung cancer in two transgenic mouse models of Raf oncogene-induced lung adenomas. The approach may provide a new strategy for the rational design of cancer therapies. Competing interests The author(s) declare that they have no competing interests. Authors' contributions IG, JF and JT designed the study. IG drafted the manuscript. JF was also involved in writing the report. AS and JF did the FACS analyses. JT constructed the EL4Raf cell line. TP, AS, JF and IG carried out the immunization of mice and the Western blot analyses. WG and URR were involved in providing the conceptual framework for this study. All authors approved the final version of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Supplementary figure 1 : C-Raf-specific IgG in sera of p.o/i.v. (A) or i.n (B) immunized BxB23 mice (serum dilution 1:1000) demonstrated by western blotting. Supplementary figure with WESTERN Blot analysis of positive C-Raf sera. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549196.xml
555583	Design considerations for a wearable monitor to measure finger posture	Background Objective measures of hand function as individuals participate in home and community activities are needed in order to better plan and evaluate rehabilitation treatments. Traditional measures collected in the clinical setting are often not reflective of actual functional performance. Recent advances in technology, however, enable the development of a lightweight, comfortable data collection monitor to measure hand kinematics. Methods This paper presents the design analysis of a wearable sensor glove with a specific focus on the sensors selected to measure bend. The most important requirement for the glove is easy donning and removal for individuals with significantly reduced range of motion in the hands and fingers. Additional requirements include comfort and durability, cost effectiveness, and measurement repeatability. These requirements eliminate existing measurement gloves from consideration. Glove construction is introduced, and the sensor selection and glove evaluation process are presented. Results Evaluation of commercial bend sensors shows that although most are not appropriate for repeatable measurements of finger flexion, one has been successfully identified. A case study for sensor glove repeatability using the final glove configuration and sensors does show a high degree of repeatability in both the gripped and flat hand positions (average coefficient of variability = 2.96% and 0.10%, respectively). Conclusion Measuring functional outcomes in a portable manner can provide a wealth of information important to clinicians for the evaluation and treatment of movement disorders in the hand and fingers. This device is an important step in that direction as both a research and an evaluation method.	Background Rehabilitation research has recently begun to emphasize the use of objective outcome measures to assess the efficacy of rehabilitation treatment or intervention [ 1 ]. These goals could be greatly furthered through the development of wearable measurement systems that provide an opportunity to evaluate how the individual participates in home and community activities. Information collected in this manner can provide a more realistic snapshot of activity and function than traditional methods which restrict measurements to the clinical or research site. Data describing actual usage in the home is especially important for the hand as hand movement is so closely tied to performance of functional tasks. In order to understand how individuals truly interact with their environments, we wish to obtain quantitative measures of finger flexion and extension over longer periods of time than traditionally investigated (such as monitoring over a full circadian cycle). Unfortunately, rehabilitation researchers have very few methods available to objectively evaluate hand use and function outside the clinic, especially for individuals with moderate to severe reduction in range of motion in the hand and fingers. Joint range of motion (ROM) is a primary measure in hand rehabilitation, and is traditionally assessed using manual goniometry. Manual methods, however, are limited to static measurements. In addition, they can be adversely affected by common issues such as inter- and intra-operator error and operator experience level [ 2 ]. In order to objectively measure hand use outside the clinic, the selected method must be both portable and capable of recording continuous streams of data over time. Automated measurement methods can meet these requirements as well as eliminate most operator-related issues. For example, 24-hour monitoring has proven useful for tracking parameters such as heart rate and blood pressure, and the same concept can be extended to other useful parameters, although currently no wearable devices are available to measure finger posture in a similar manner. Practical medical applications can include providing input for virtual reality therapy, measuring hand function in the planning of rehabilitation therapies, or evaluating the outcome of interventions under more realistic conditions. Sensor gloves have been proposed to provide automatic measurements of finger and joint position during different activities, with the virtual reality industry continuing to drive the market for sensor gloves [ 3 ]. For example, Fifth Dimension Technologies (5DT, Irvine, CA) produces a 5-sensor and a 16-sensor version wireless sensor glove which transmits data to a nearby computer. Two joints per finger are captured with these expensive devices (proximal interphalangeal joint (PIP) and metacarpophalangeal joint (MCP)) using fiber optic sensors. The White Hand Group (Mississippi State University) is developing a lower-cost (<$500) flexion data glove using fiber optics that has two sensors per finger. The glove is tethered to a computer and is aimed at VR and gaming applications rather than accurate measurement applications. CyberGlove (Immersion Corporation, San Jose, CA) contains 5 to 22 "patented bend-sensing technology" strain gauges to measure individual joint movements. This glove, however, is very expensive and difficult for stroke survivors to don. The Essential Reality (Mineola, NY) P5 glove also uses bend sensors, Abrams/Gentile flex sensors (wired flexion measurements), and infrared (IR) emitters (line-of-site wireless position and rotation measurements). The flex sensors are attached to each finger by rings between the proximal and distal interphalangeal joints (DIP). While price of this glove is appealing (<$200), the glove is not portable and requires the wearer to keep the top of the glove always facing a fixed antenna IR receiver. The Humanglove™ (Humanware S.R.L., Pisa, Italy) is a flexible glove with 20 Hall effect sensors to measure bend. The Humanglove was evaluated for feasibility and repeatability in finger range of motion in all joints; work continues to establish the measurement accuracy [ 2 ]. Several research gloves have been reported with no clinical results. Karlsson et al. [ 4 ] determined finger flexion by measuring the pressure changes in airtight polyvinyl tubes on three fingers. Zurbrügg [ 5 ] measured flexion using potentiometers mounted on the back of the hand, although the construction is not durable for long term measurements. Hofmann and Henz [ 6 ] used inductive length encoders attached to a cotton glove to measure flexion and gestures in real time. The glove is not easy to put on, and the sensors can move around relative to the joint position. Jurgens et al. [ 7 ] proposed an innovative method using electrically conducting ink printed on a flexible polyester plastic for a low cost solution, although disadvantages include extreme sensitivity to small changes in temperature, and a moderately slow response time. The existing glove systems do not meet the needs set forth in our device requirements. Although some gloves are lightweight, others such as force feedback and exoskeleton based gloves are too heavy and bulky for home use by individuals with hand impairments. Most are too expensive or require custom sizing to reduce errors; measurement errors are decreased when gloves fit more snugly [ 2 ]. However, donning a tight glove can be difficult to impossible for some individuals with movement disorders in the hand. Full gloves have other limitations as well. Actions such as wrist flexion and rotation in full fabric gloves can cause the glove material to move over the skin. In this case, friction can prevent the material from returning completely to the original position, leaving the sensors located in different positions over the joints [ 2 ]. This source of error is manifested as a drift in measured bend that is difficult to detect, characterize, and eliminate from recorded data streams. The environment proposed for the glove use is also very challenging. A majority of the existing methods are not appropriate for wearable measurements in the home and community, and none are designed for use with clinical and rehabilitation populations who may exhibit significant range of motion restriction in the fingers. The long term goal of this project, therefore, is to design, build, and clinically evaluate a novel low-cost wireless device to measure hand and finger activity while individuals participate in normal home and community activities. It will be used to study the loss of hand function that can occur following neurological disorders such as stroke and traumatic brain injury, and to evaluate how treatments can improve an individual's ability to function in the home and community environments. This article describes the design process for the development of the glove, discussing wearability issues such as comfort, durability and weight and focusing on sensor characterization and selection. The results of an initial evaluation of measurement repeatability while wearing the glove are presented. Appropriate characterization of the glove must occur in two phases: evaluation of the sensors separately, and then evaluation of the entire glove after appropriate sensors have been identified and characterized. Full repeatability results and measurement accuracy will be reported in the future. Methods The creation of effective custom measurement systems requires detailed attention to the requirements and design analysis phases of the development process. While it may be tempting to solve multiple problems with one system, this often leads to overly complicated devices that take too long to complete, and may not actually meet the core requirements. To avoid this scenario, the sensor glove project focused specifically on a set of core requirements presented below, and pursued a multi-step analysis of the design to ensure the requirements were being appropriately addressed. The steps include an analysis of glove construction methods, characterisation of sensors, and exploration of sensor repeatability and accuracy on the bench and during subject trials. The primary requirements fall into four categories. 1) Donning and Removing: The glove must be easy to don and remove for individuals having reduced range of motion in the hand and fingers. 2) Comfort and Durability: The glove must be lightweight and unobtrusive, and permit comfortable wearing for at least 24 hours. It must not restrict range of motion or represent a snag hazard during use. 3) Function: The glove must detect a wide range of activities, including fine motor activities such as writing. Measurements must be accurate and repeatable. The sensors must not move around, but remain in the same position with respect to the joints and phalanges over time. The system must allow the performance of normal daily activities, although use in water is not required. Measurements must be repeatable with an error no more than 5% of full scale. 4) Cost: Manufacturing cost is an important consideration for several reasons. An inexpensive device allows several to be deployed simultaneously to perform research studies more quickly. A low-cost glove can be considered disposable for sanitary reasons. Finally, wearers may unknowingly limit or modify their hand motions and activities in an attempt to protect an expensive device. A low-cost alternative ensures that more realistic and representative data are captured and recorded. As noted, a variety of sensors have been employed to measure joint angle, including strain gauges or bend sensors, fiber optics, pneumatics, or Hall effect sensors. While each has advantages and disadvantages, the requirements for our glove preclude the use of all but the bend sensors. Using fiber optics to measure bend requires a light source such as a light emitting diode and a photo detector. The amount of bend is proportional to the attenuation of detected light in specially treated sections of fiber that pass over the tops of the finger joints. Disadvantages of this method include the complexity of glove construction and price. Hall effect sensors, which detect magnetic fields, and can be configured as proximity sensors to provide a linear output proportional to distance from a magnetic source. By placing a series of sensors on the back of a glove in a predefined pattern, the joint angle can be computed from the changing field strengths measured by the sensors when the fingers bend. While these devices are small, the resulting glove can be somewhat bulky and will require a magnetic source, adding to overall weight. Interference from other electromagnetic sources is also a concern. Strain gauges detect stretch produced by joint rotation. They may have very high accuracy, but are expensive and often delicate. Bend sensors offer a lightweight and inexpensive alternative. These sensors are thin flexible membranes that change resistance when bent; increasing bend angle is generally associated with increased measured resistance. One or more layers of carbon and a conductive material are applied over a thin substrate. Depending on the sensor type, bending motion forces conductive particles further apart, increasing the resistance to current flow. These sensors are popular for detecting obstacles and measuring large changes in bend angle, and are proposed for accurate measurement of finger posture. However, most exhibit a time-varying creep behavior when held in a fixed bent position that reduces the accuracy of measurement. We sought to find bend sensors that would be feasible. Glove Construction Methods In order to explore the first two requirements, ease of donning and comfort/durability, several prototype glove systems were created in order to identify the best materials and construction methods to satisfy these requirements. Several materials were evaluated, including blends of Lycra ® , Nylon and cotton. The material must exhibit stretch so that finger motion and bending are not restricted. While some blends resisted finger motions, most were flexible enough that the wearer could forget the glove was on. A discussion of the process and final selection of glove materials and application method can be found elsewhere [ 8 ]. The final glove material is a 93% Lycra ® / 7% Nylon blend; it is used to create thin sleeves into which the sensors are inserted. One sleeve is attached to the back of each finger in order to locate the sensor directly over each joint; the optimal adhesive is very thin, double-sided toupee tape. Applying the sensors to the back of the fingers, rather than using a traditional glove that must be donned, allows easy application and removal for individuals who cannot fully open all fingers to put on a traditional glove. Total glove cost without sensors is less than $2.50. Comfort and durability were evaluated over a 24 hour period. The glove survived intact and did not impede any activities other than showering and tucking in a shirt. Because the sensors are attached to the back of the hand, the palmar surface of the hand is uncovered and free of obstruction, leaving the sense of touch intact. While only one individual was used to narrow down the different prototype ideas, 24 individuals (12 with brain injury and 12 healthy controls) are currently participating in a study to more fully evaluate the glove configuration and performance. Sensor Repeatability An early decision was made to use an inexpensive bend sensor as the sensing element for its low profile, light weight, and low cost. While the first prototype glove has only 5 sensors, the design provides the flexibility to add additional sensors for all joints and for finger adduction/abduction. Sensors from several manufacturers were characterized in order to determine if measurements were repeatable and if large changes in finger posture and fine motor control could be captured. In addition, the calibration relationship between bend angle and measured resistance was evaluated. The importance of repeatability testing cannot be overemphasized. The sensors were evaluated separately before being incorporated into the sensor glove. Two types of tests were performed using a set of tubes of known diameter: 1) determination of full scale and the resistance-bend relationship (using tube diameters: 4", 3", 2", and 1.5", 1" and 0.75"), and 2) evaluation of measurement repeatability (using the 3" diameter tube). A physical guide was placed on each tube to ensure that the sensor was placed in the same location on the tube each time it was applied. From four to ten sensors from each manufacturer were evaluated. To test the sensors, each was initially placed flat on the table for several seconds, then bent over a single calibration tube for 30 to 60 seconds, and then placed flat again. Resistance readings were taken during each phase. The expected profile for this activity should look like a rectangular wave between two resistance values: a lower resistance value in the flat position, then the higher value when bent on the tube, and ending with the original lower flat value. Multiple measurements were taken to evaluate the variation in sensor outputs, and to construct a bend resistance versus tube diameter relationship that could be used for calibration purposes. Full scale is determined from the endpoints of the calibration relationship. The minimum measured resistance value corresponds to the flat position, and the maximum resistance value is represented by the resistance measured on the 0.75" calibration tube. The maximum value is an estimate of what would be observed in human subjects because the bending radius over a finger joint is not exactly replicated by bending over different diameter tubes; however, making this assumption allows the different sensors to be compared to one another for selection purposes. Full scale error is computed as the percent change in resistance measured while a sensor is fixed unmoving on a calibration tube with respect to the full scale range. A second error is also reported, and is calculated as the percent change in the peak sensor resistance with respect to the magnitude of the step function rise in resistance when the sensor is positioned on a calibration tube. This error calculation was also selected for comparison because a number of everyday activities such as grasping and moving objects find the hand in roughly this position. As discussed in the results section, additional analysis was performed to explore an unexpected time-varying behavior of the sensors. Collection of these data and of the data described in the next section was performed using a host computer with an 8 channel 16-bit A/D card. The sensors were connected to an interface box and data on all sensors was collected at 100 Hz using LabView (National Instruments, Austin, TX). Analysis was performed using Microsoft Excel. Sensor Glove Repeatability A major concern in developing a measurement method is that measurements are repeatable. If the bend sensors and the configuration of these sensors on the fingers will be used for several measurements during the same session, or for measurements over several sessions, then repeatability must be established before the data can be given credence. Rigorous validation of repeatability, however, is often lacking from descriptions of various "data gloves." One method to evaluate repeatability of sensor glove-type devices has been proposed by Wise [ 9 ] and expanded by Dipietro [ 2 ]. The method was specifically developed for devices that perform semi- or fully-automated goniometric measurements. Repeatability was established using a custom mold created by each subject. The mold was made while the subject simulated a grip position around a wet mixture of Plaster of Paris. After the mold dried, it was used to ensure that the subject's finger positions were identical for several consecutive gripping actions on the mold. Testing with 5 healthy control subjects revealed errors that led to recommendations for improvements in measurement methodology. Dipietro [ 2 ] repeated these procedures with some clarifications in hand position in the evaluation of the Humanglove, and echoed the need for standardized testing protocols for sensor glove devices. This standardized repeatability testing protocol procedure includes four tests. We will use two, Tests A and C, which focus on repeatability of multiple measurements over a single data collection session. Tests A and C are performed here because the first focus of the glove is for single data collection sessions (which can last up to 24 hours). The protocol used here follows the modifications proposed in [ 2 ]; an overview of the tests appears below. Test A: A roughly cylindrical plaster mold is custom created for each subject to ensure that the fingers are flexed to the same position for each test. The participant clenches the mold for 6 seconds and then releases the mold for 6 seconds. This clench/release cycle is repeated 10 times. Repeatability measurements are taken from each sensor during the clench phases. Test C: The participant places the hand on a table top and alternately raises the hand and lightly flexes the fingers, and returns the hand to the table top for 6 second each. Repeatability of the flat hand position is explored in this test. In order to achieve repeatability in hand and finger position, and outline of the hand profile is drawn on paper and placed on the table. This cycle is also repeated 10 times. For each test above, the participant rested for at least 1 minute, and then repeated the entire test. This was done 10 times for both Test A and Test C, for a total of 100 grip/release cycles for each test. Descriptive statistics are computed (mean, standard deviation, and coefficient of variation). The percent coefficient of variation (standard deviation divided by the mean*100%) is used to compare the measurement variability among the five digits and between the two repeatability tests. Results and Discussion Glove Construction Figure 1 shows a prototype of the sensor glove monitor. For the test shown here, one sensor was used to measure flexion of each metacarpophalangeal (MCP) joint. The sensors are located inside the beige sensors sleeves, which are attached to the back of the metacarpals and proximal phalanges. The sensors do not move relative to the joint under measurement. The forearm-mounted box contains signal conditioning. In the next prototype, the box will also contain a wireless transmitter and the cable from the left of the box will be removed, allowing the participant to move around freely. Instead of Velcro bands, a comfortable band of flexible material will hold the box to the forearm. Data will initially be transmitted wirelessly to a nearby laptop computer, and eventually transmitted wirelessly to a waist mounted data recorder. The sensors and glove sleeves weigh less than 7.1 grams; adding the signal conditioning box increases the weight to 85 grams. The final device will have the added weight of a battery and small wireless transmitter. Figure 1 Prototype of the sensor glove monitor. The monitor is shown with five sensors placed on the MCP joints. Signal conditioning is contained in the box. The next prototype will include a wireless link for data download to an external computer, enabling the removal of the cable extending out of the back of the box. Sensor Repeatability Performing sensor evaluation and characterization early in the design process allowed us to identify several shortcomings of commercial bend sensors and eventually select an appropriate sensor. The first sensor evaluated was the Abrams-Gentile Entertainment, Inc. (New York, NY) sensor patent #5,086,785. Attempts to measure repeatable bend resistance versus calibration tube diameter failed because the measured resistance decayed over time. The Abrams-Gentile sensor exhibits the most common behavior that we will refer to as Type A behavior, and it appears in Figure 2 as line "AG". The sensor reached a peak resistance value just as it was wrapped around the calibration tube, with an immediate decay in resistance over time. We expected that the sensor values would be constant; however, the drift in measured resistance prevented an accurate and repeatable measurement of bend. To eliminate other potential sources of error, the analysis was repeated on ten other sensors, and the problem finally isolated to the sensors by testing each directly using an ohmmeter. Figure 2 Sensor time-varying properties. Each of several sensors is placed on a calibration tube for at least 30 seconds, and then stretched flat on a table in order to verify that a constant relationship exists between bend angle and measured resistance. Ideally, these curves should be flat but a significant time-varying decay renders most unusable for this application. This figure shows several representative curves for the three manufacturers evaluated. AG: Abrams-Gentile sensors, SS: SpectraSymbol sensors, FP: Flexpoint sensors (several types). The average decay in resistance while on the tube was computed. After 30 seconds, the average error for the Abrams-Gentile sensors was 9.5% of full scale, and 24.4% of step function rise resistance (Table 1 ). The Abrams-Gentile sensor never settled on a final resistance value, but over an extended two-day data collection session continued to slowly decay. While these sensors are appropriate for many applications such as position detectors and indicators of gross movement, we determined that they are not appropriate for accurate and repeatable measurements of finger flexion. Table 1 Sensor decay over time for three sensor types fixed over a 3-inch tube % Full Scale % Step Function Rise Time (sec) Abrams-Gentile Flexpoint Time (sec) Abrams-Gentile Spectra Symbol Flexpoint 0 0.0% 0.0% 0 0.0% 0.0% 0.0% 2 3.9% 0.3% 2 9.9% 15.2% 2.6% 4 5.2% 0.3% 4 13.4% 17.8% 3.2% 15 7.9% 0.6% 15 20.4% 25.7% 6.1% 30 9.5% 0.8% 30 24.4% 31.8% 8.9% The same testing was repeated using sensors from Spectra Symbol (Salt Lake City, UT). Similarly, the step function rise in resistance measured on application to the 3" calibration ring dropped 31.8 % in the first 30 seconds (Figure 2 , Type A: SS). Again, the sensor is better suited to sensing a change in angle, rather than the magnitude of the change. A calibration relationship was not explored because the magnitude of the error was so large. Six different sensor configurations were evaluated from Flexpoint (South Draper, UT). These included flex sensors with an overlaminate adhered by a pressure sensitive adhesive (sensor #1), with a robust polyimide overlaminate (sensor #2), with no overlamination but with a stiff backer (sensor #3), and an overmolded sensor (sensor #4) for harsh environmental conditions. Representative contours for the 3" calibration test are shown in Figure 2 , labelled "FP: #1, 2, 4, and 5" Sensor 3 exhibited the same large decays observed with the Type A Abrams-Gentile and SpectraSymbol sensors shown in the figure. In contrast, sensors 1 and 2 (Type B) responded to the initial fast bend over the tube with a slow rise in resistance that never reached a peak. When the sensors were removed from the tube and placed flat again, the resistance decayed but did not reach baseline values for many minutes. Sensor 4 (Type C) exhibited a fast response to a peak value, dropped 15% of the rise amount and then slowly recovered the 15% over several minutes, but never returned to the baseline value when placed flat at the end of the test. None of these sensors (1–4) is appropriate for the sensor glove. However, in consultation with the company describing our exact needs, a solution was identified. The bend sensors are generally not supplied without some type of protection layer. However, these layers tended to cause the observed decay problems, making these sensors inappropriate for this application. Evaluation of bare sensors (Figure 2 , Type D) revealed the initial peak resistance followed by decay; however, the magnitude of the decay after 30 seconds was only 0.8% full scale or 8.9% of step function resistance rise, which is acceptable for this application. The bare version of the sensor is approximately $7.10 in low quantities. The average relationship between bend angle and resistance for 5 sensor trials is shown in Figure 3 . The error results from all sensors are shown in Table 1 . Figure 3 Resistance – bend relationship of the Flexpoint sensor. The Flexpoint sensor has a nonlinear relationship between measured resistance and bend diameter, as found by measuring resistance for sensors wrapped around calibration tubes of different diameters. For illustration purposes, the relationship presented here is an average of several sensors; a separate relationship will be measured for each sensor used in the sensor glove. Bend sensors are used in a number of university and home projects, despite our findings that most are not repeatable for moderate to fine resolution measurements. Instead, most are appropriate for binary ON/OFF applications, or applications that do not require high resolution or highly repeatable results. Examples include using the sensor as a "whisker" to sense the proximity of an object for collision detection, for detecting large changes in bend angle, or for more unique applications such as adding effects to music [ 10 ]. Others report early results for such implementations as measurement of foot flexion for biofeedback [ 11 ] although follow-up work on calibration and analysis methods is still pending. We located no references validating and using these sensors, and only one reference that indicated that the Abrams-Gentile sensor was "difficult to work with" [ 12 ]. For the measurement device described here, repeatability of measurement is an important criterion. Only the bare Flexpoint sensor was found to be appropriate for measuring fine changes in bend angle in a repeatable manner. If another sensor is used in this application, there is no way to determine the actual bend radius because a wide range of measured resistance values (caused by the decay) correspond to a single bend radius. Sensor Glove Repeatability Repeatability testing began with the evaluation of the sensors and final sensor selection, and continues by considering the performance of the entire glove. The sensor glove repeatability testing has been performed with one participant, who is the first in a study that will include repeatability testing for 6 healthy controls. All participants complete an Institutional Review Board consent form and the required HIPAA authorization forms. The plaster mold was created as shown in Figure 4 . Figure 4 Grip mold. The grip mold is custom made for each subject. It provides a repeatable position for the fingers to assume for multiple grip-release activities in order to evaluate repeatability of measurement. The results of Test A reveal the repeatability of measurement in the grip hand position. Coefficient of variability for all five digits is less than 6% (thumb: 0.55%, index: 5.37%, middle: 1.91%, ring: 4.61%, pinkie: 2.36%). Figure 5 shows the mean and standard deviation of measured MCP joint position while gripping the mold 100 times. The mean values indicate the average resistance of the sensors when the fingers are gripping the mold. In this test, the actual mean value of bend is not critical, it just represents the joint position when the mold was made. In addition, these values are not calibrated. For this individual, the thumb MCP is the least bent (having the lowest resistance values) while the ring and pinkie fingers are significantly bent. The repeatability information is located within the very low coefficient of variation, or variation of the measured values about the mean. Figure 5 Repeatability testing of grip position. Repeatability testing of one participant for the grip test (Test A). Means and standard deviations are shown. Mean values differ because each finger is in a different position when gripping the mold. Repeatability information is contained in the variation around the mean. The results for Test C show the repeatability in the flat hand position with the sensors fully extended. Coefficient of variability for all five digits is less than 1% (thumb: 0.18%, index: 0.08%, middle: 0.05%, ring: 0.07%, pinkie: 0.15%). Figure 6 shows the mean and standard deviation of measured MCP joint position while placing the hand flat 100 times. Descriptive statistics and coefficient of variability for Tests A and C are shown in Table 2 . The variation in measurements for the flat hand position is extremely small over the 100 cycles, which is very encouraging considering that the flat hand position is only guided by an outline of the hand on the tabletop. Five additional participants will complete this repeatability testing and results presented in the future. These results are similar to [ 2 ] in that the measurement repeatability was better in the flat hand (Test C) case than in the grip mold case (Test A). Dipietro [ 2 ] speculated that the "hand is positioned more accurately by placing it flat...than by clenching the mold." Wise [ 9 ] also noted that "increasing forces produced errors in the glove measurements, especially in the MCP joints." We observed through separate experimentation that in the flat hand position, the musculature in the hand tends to relax. In the grip position, in contrast, the muscles must maintain at least a minimal contraction in order to prevent dropping the mold. Varying levels of grip force can be applied, and the finger positions can be shifted slightly while still holding the mold closely. The measured variations in resistance easily accounted for the observed variations in the grip repeatability test results. The solution was to ask the individual to only grip the mold with enough strength to hold it, and that instruction will be given to future participants. While executing these tests can be challenging, we must concur that standardized testing is vital to ensuring that the collected data are useful and repeatable. Conclusion The glove developed in this research is unconventional, and its uniqueness owes to the appropriate attention to the core requirements during the design analysis phase. The glove provides a novel method to evaluate actual functional capacity, starting with the dynamic evaluation of ROM as individuals participate in their normal daily activities. The glove is not a generic solution, but a specific device to measure finger posture in an underserved population. Bend sensors were selected for their lightweight low profile, and for cost effectiveness. Although significant error can be introduced by using bend sensors, sensors with the appropriate repeatability characteristics have been identified. The bare Flexpoint sensors provided repeatable measurements with a 30 second error of 0.8% full scale, as compared to 9.5% for the next best solution, the Abrams-Gentile sensor. The overall glove configuration shows strong promise for providing repeatable measurements over long periods of time without undesired movement over the joints. Coefficient of variability averaged 2.96% and 0.10% across the five digits for the grip test and flat hand test, respectively. The glove is low cost; the total cost for the disposable portion of the device (glove material, adhesive and sensors) is less than $40, which is significantly less than any other reported solution. The glove can be used not only to measure flexion in individuals with reduced range of motion who cannot wear traditional measurement gloves, but also to measure passive ROM and cleaning activities in individuals who cannot initiate volitional activity in their affected hand. Future directions include completion of the current study to establish repeatability and to identify calibration methods. The wireless link will be added midway through the study to provide full wearability and unencumbered movement, paving the way for extended studies in the home and community environments. Competing interests The author(s) declare that they have no competing interests. Authors' contributions LS made substantial contributions to the conception and design of the device and drafted the manuscript. DK identified a need for the device, contributed to the requirements and design of the device, and participated in revisions of the manuscript. Figure 6 Repeatability testing of flat hand position. Repeatability testing of one participant for the flat hand test (Test C). Means and standard deviations are shown. Mean values are similar because all fingers are straight when data collection occurs. Table 2 Variability in glove measurements for repeatability Tests A and C Test A: Grip Mold Test C: Flat Hand Mean (Ohms) SD (Ohms) CoV Mean (Ohms) SD (Ohms) CoV Thumb 9138 50 0.55% 8257 15 0.18% Index 14400 773 5.37% 8562 7 0.08% Middle 27873 533 1.91% 7992 4 0.05% Ring 51364 2368 4.61% 8231 6 0.07% Pinkie 50629 1194 2.36% 7586 11 0.15%	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555583.xml
522750	Overview of the diagnostic value of biochemical markers of liver fibrosis (FibroTest, HCV FibroSure) and necrosis (ActiTest) in patients with chronic hepatitis C	Background Recent studies strongly suggest that due to the limitations and risks of biopsy, as well as the improvement of the diagnostic accuracy of biochemical markers, liver biopsy should no longer be considered mandatory in patients with chronic hepatitis C. In 2001, FibroTest ActiTest (FT-AT), a panel of biochemical markers, was found to have high diagnostic value for fibrosis (FT range 0.00–1.00) and necroinflammatory histological activity (AT range 0.00–1.00). The aim was to summarize the diagnostic value of these tests from the scientific literature; to respond to frequently asked questions by performing original new analyses (including the range of diagnostic values, a comparison with other markers, the impact of genotype and viral load, and the diagnostic value in intermediate levels of injury); and to develop a system of conversion between the biochemical and biopsy estimates of liver injury. Results A total of 16 publications were identified. An integrated database was constructed using 1,570 individual data, to which applied analytical recommendations. The control group consisted of 300 prospectively studied blood donors. For the diagnosis of significant fibrosis by the METAVIR scoring system, the areas under the receiver operating characteristics curves (AUROC) ranged from 0.73 to 0.87. For the diagnosis of significant histological activity, the AUROCs ranged from 0.75 to 0.86. At a cut off of 0.31, the FT negative predictive value for excluding significant fibrosis (prevalence 0.31) was 91%. At a cut off of 0.36, the ActiTest negative predictive value for excluding significant necrosis (prevalence 0.41) was 85%. In three studies there was a direct comparison in the same patients of FT versus other biochemical markers, including hyaluronic acid, the Forns index, and the APRI index. All the comparisons favored FT (P < 0.05). There were no differences between the AUROCs of FT-AT according to genotype or viral load. The AUROCs of FT-AT for consecutive stages of fibrosis and grades of necrosis were the same for both moderate and extreme stages and grades. A conversion table was constructed between the continuous FT-AT values (0.00 to 1.00) and the expected semi-quantitative fibrosis stages (F0 to F4) and necrosis grades (A0 to A3). Conclusions Based on these results, the use of the biochemical markers of liver fibrosis (FibroTest) and necrosis (ActiTest) can be recommended as an alternative to liver biopsy for the assessment of liver injury in patients with chronic hepatitis C. In clinical practice, liver biopsy should be recommended only as a second line test, i.e. , in case of high risk of error of biochemical tests.	Background One of the major clinical problems is how to best evaluate and manage the increasing numbers of patients infected with the hepatitis C virus (HCV) [ 1 ]. Liver biopsy is still recommended in most patients [ 2 , 3 ]. However, numerous studies strongly suggest that due to the limitations [ 4 - 6 ] and risks of biopsy [ 7 ], as well as the improvement of the diagnostic accuracy of biochemical markers [ 8 , 9 ], liver biopsy should no longer be considered mandatory. Among the non-invasive alternatives to liver biopsy [ 10 ], several studies have demonstrated the predictive value of two combinations of simple serum biochemical markers in patients infected with HCV: FibroTest (FT; Biopredictive, Paris, France; HCV-Fibrosure, Labcorp, Burlington, USA) for the assessment of fibrosis; and ActiTest (AT; Biopredictive, Paris, France) for the assessment of necroinflammatory activity (necrosis) [ 8 , 9 , 11 - 21 ]. Similar results have not been obtained with other diagnostic tests [ 10 - 17 ]. Since September 2002 these tests (FT-AT) have been used in several countries as an alternative to liver biopsy. In a recent systematic review, it was concluded that these panels of tests might have the greatest value in predicting fibrosis or cirrhosis [ 10 ]. It was also stated that biochemical and serologic tests were best at predicting no or minimal fibrosis and at predicting advanced fibrosis/cirrhosis, and were poor at predicting intermediate levels of fibrosis [ 10 ]. The aim of this study was to summarize the diagnostic value of these tests by an overview of the scientific literature and to respond to the following frequently asked questions by performing original new analyses: 1) what is the range of the FT-AT diagnostic values across the different studies? 2) What are the base evidence comparisons between FT-AT and other published biochemical markers? 3) Are there differences in diagnostic values according to HCV genotype or viral load? 4) Are there differences between the FT-AT diagnostic values according to stages and grades? – In other words, is FT better at predicting no or minimal fibrosis (F0 vs F1) or advanced fibrosis/cirrhosis (F3 vs F4) than at predicting intermediate levels of fibrosis (F1 vs F2)? And 5) what is the conversion between FT-AT results and the corresponding fibrosis stages and necrosis grades? Results Analysis of the literature Between February 2001 and March 2004, a total of 16 publications [ 8 , 9 , 11 - 21 , 24 - 26 ] and 4 abstracts [ 27 - 30 ] without corresponding publications were identified. Diagnostic value of FT-AT among published studies For 12 groups of patients detailed in 6 publications [ 8 , 11 , 12 , 14 , 19 , 26 ], it was possible to assess the prevalence of significant fibrosis and the FT area under receiver operating characteristics curve (AUROC) values, as well as the sensitivity and specificity for the 4 different FT cut offs (Table 1 ). For the diagnosis of significant fibrosis by the METAVIR scoring system, the AUROC ranged from 0.73 to 0.87, significantly different from random diagnosis in each study (Table 1 ), in meta-analysis (mean difference in AUROC = 0.39, random effect model Chi-square = 529, P < 0.001) (Figure 1 , upper panel), or after pooling data in the integrated database (Table 2 ). For the cut off of 0.31, the FibroTest negative predictive value for excluding significant fibrosis (prevalence 0.31) was 91% (Table 2 ). Table 1 Summary of the diagnostic value of FibroTest for the staging of hepatic fibrosis and comparisons with hyaluronic acid, the Forns Index and the APRI Index in patients with chronic hepatitis C, from the published studies. First author N* Methodology Marker Stage/Prevalence AUROC SE Cut off Sensitivity Specificity Imbert-Bismut, 2001 189 Prospective Single center First year cohort FibroTest F2F3F4 / 0.38 0.84 (0.03) 0.10 0.30 0.60 0.80 0.97 0.79 0.51 0.29 0.24 0.65 0.94 0.95 Imbert-Bismut, 2001 134 Prospective Single center Validation cohort FibroTest F2F3F4 / 0.45 0.87 (0.03) 0.10 0.30 0.60 0.80 1.00 0.87 0.70 0.38 0.22 0.59 0.95 0.97 Poynard, 2001 165 Retrospective Randomized trial Multicenter FibroTest F3F4 Knodell / 0.32 0.74 (0.03) 0.10 0.30 0.60 0.80 0.96 0.81 0.50 0.13 0.24 0.65 0.92 0.98 Poynard, 2001 165 Retrospective Randomized trial Multicenter Hyaluronic F3F4 Knodell / 0.32 0.65 (0.03) 20 40 100 0.81 0.47 0.23 0.39 0.65 0.91 Poynard, 2003 352 Retrospective Randomized trial Multicenter Before treatment FibroTest F2F3F4 / 0.39 0.73 (0.03) 0.10 0.30 0.60 0.80 0.97 0.86 0.50 0.20 0.08 0.45 0.79 0.95 Poynard, 2003 352 Retrospective Randomized trial Multicenter After treatment FibroTest F2F3F4 / 0.32 0.77 (0.03) 0.10 0.30 0.60 0.80 0.98 0.85 0.46 0.16 0.15 0.39 0.81 0.97 Rossi, 2003 125 Prospective Multicenter Non-validated analyzers FibroTest F2F3F4 / 0.38 0.74 (0.05) 0.10 0.30 0.60 0.80 0.92 0.75 0.42 0.22 0.29 0.61 0.94 0.96 Myers, 2003 130 Retrospective Single center HCV-HIV Co-infection FibroTest F2F3F4 / 0.45 0.86 (0.04) 0.10 0.30 0.60 0.80 0.98 0.90 0.66 0.34 0.17 0.60 0.92 0.96 Thabut, 2003 249 Retrospective Single center From Imbert-Bismut, 2001 FibroTest F2F3F4 / 0.38 0.84 (0.02) 0.10 0.30 0.60 0.80 0.98 0.84 0.58 0.29 0.22 0.65 0.93 0.95 Thabut, 2003 249 Retrospective Single center From Imbert-Bismut, 2001 Forns Index F2F3F4 / 0.38 0.78 (0.03) 1 3 6 8 1.00 1.00 0.55 0.19 0.04 0.26 0.86 0.97 Le Calvez, 2004 323 Retrospective Single center From Imbert-Bismut, 2001 FibroTest F2F3F4 / 0.41 0.83 (0.02) 0.10 0.30 0.60 0.80 0.97 0.81 0.58 0.33 0.30 0.66 0.93 0.95 Le Calvez, 2004 323 Retrospective Single center From Imbert-Bismut, 2001 APRI Index F2F3F4 / 0.41 0.74 (0.03) 0.50 1.00 1.50 2.00 0.81 0.54 0.36 0.24 0.56 0.84 0.91 0.95 Callewaert, 2004 82 Prospective FibroTest F4 / 0.29 0.89 (0.04) 0.10 0.30 0.60 0.80 1.00 0.92 0.79 0.67 0.33 0.62 0.81 0.92 Callewaert, 2004 82 Prospective Glyco Cirrho Test F4 ** / 0.29 0.87 (0.04) -0.2 0.1 0.4 0.6 1.00 0.79 0.21 0.17 0.12 0.88 0.95 1.00 * Number of patients. ** Compensated Figure 1 Meta-analysis of the AUROC observed in published studies of FibroTest diagnostic value. AUROCs were all significantly higher for FibroTest than the random 0.50 value (upper panel) (P < 0.001). AUROCs of FibroTest were significantly higher then AUROCs of other fibrosis markers (lower panel) (P < 0.05). Table 2 Integrated database, with predictive values for significant hepatic fibrosis according to METAVIR conversion cut offs. Derived from published studies. Integrated database Patient number Marker Stage/Prevalence AUROC (SE) Cut off used for METAVIR stages conversion Sensitivity Specificity Negative predictive value Positive predictive value With Blood Donors 1,570 FibroTest F2F3F4/0.31 0.83 (0.01) 0.21 0.92 0.55 0.94 0.48 0.27 0.87 0.62 0.92 0.51 0.31 0.84 0.68 0.91 0.54 0.48 0.68 0.81 0.85 0.61 0.58 0.56 0.87 0.82 0.67 0.72 0.38 0.95 0.77 0.76 0.74 0.35 0.95 0.76 0.76 0.75 0.33 0.96 0.76 0.78 Without blood donors 1,270 FibroTest F2F3F4/0.38 0.78 (0.01) 0.21 0.92 0.41 0.89 0.49 0.27 0.87 0.48 0.86 0.51 0.31 0.84 0.55 0.85 0.54 0.48 0.68 0.73 0.79 0.61 0.58 0.56 0.83 0.75 0.67 0.72 0.38 0.95 0.70 0.76 0.74 0.35 0.93 0.70 0.76 0.75 0.33 0.94 0.69 0.78 For four groups of patients detailed in two publications [ 8 , 11 ], it was possible to assess the prevalence of significant necrosis and the AT AUROC values, as well as the sensitivity and specificity for 4 different AT cut offs (Table 3 ). For the diagnosis of significant necrosis by the METAVIR scoring system, the AUROC ranged from 0.75 to 0.86, significantly different from random diagnosis in each study (Table 3 ), in meta-analysis (mean difference in AUROC = 0.29, random effect model Chi-square = 556, P < 0.001), or after pooling data in the integrated database (Table 4 ). For the cut off of 0.36, the ActiTest negative predictive value for excluding significant necrosis (prevalence 0.41) was 85% (Table 2 ). Table 3 Summary of the diagnostic value of ActiTest for the diagnosis of necroinflammatory hepatic activity (AUROC) in patients with chronic hepatitis C, from the published studies. First author, Year Patient number Methodology Marker Grade/Prevalence AUROC (SE) Cut off Sensitivity Specificity Imbert-Bismut, 2001 189 Prospective Single center ActiTest A2A3 / 0.33 0.79 (0.03) 0.10 0.30 0.60 0.80 0.99 0.91 0.70 0.49 0.07 0.42 0.75 0.88 Imbert-Bismut, 2001 134 Prospective Single center Validation cohort ActiTest A2A3 / 0.28 0.75 (0.03) 0.10 0.30 0.60 0.80 1.00 0.94 0.67 0.42 0.07 0.33 0.65 0.87 Poynard, 2003 352 Retrospective Randomized trial Multicenter Before treatment ActiTest A2A3 / 0.83 0.75 (0.03) 0.10 0.30 0.60 0.80 1.00 0.90 0.49 0.20 0.00 0.38 0.87 0.99 Poynard, 2003 352 Retrospective Randomized trial Multicenter After treatment ActiTest A2A3 / 0.39 0.86 (0.02) 0.10 0.30 0.60 0.80 0.91 0.75 0.38 0.14 0.59 0.83 0.98 0.996 Table 4 Integrated database, with predictive values for the diagnosis of significant necroinflammatory hepatic activity according to METAVIR conversion cut offs. Derived from published studies. Integrated database Patient number Marker Grade/Prevalence AUROC (SE) Cut off used for METAVIR stages conversion Sensitivity Specificity Negative predictive value Positive predictive value With Blood Donors 1,570 ActiTest A2A3/0.41 0.85 (0.01) 0.17 0.95 0.55 0.94 0.60 0.29 0.87 0.69 0.88 0.66 0.36 0.81 0.74 0.85 0.69 0.52 0.62 0.86 0.76 0.75 0.60 0.51 0.90 0.72 0.77 0.61 0.50 0.90 0.72 0.78 0.62 0.49 0.91 0.72 0.78 Without blood donors 1,270 ActiTest A2A3/0.51 0.78 (0.01) 0.17 0.95 0.40 0.89 0.62 0.29 0.87 0.55 0.80 0.67 0.36 0.81 0.63 0.76 0.69 0.52 0.62 0.79 0.67 0.75 0.60 0.51 0.85 0.63 0.77 0.61 0.50 0.85 0.62 0.78 0.62 0.49 0.86 0.62 0.78 Comparison of FT-AT diagnostic values with other biochemical markers In four studies there was a direct comparison in the same patients of FT versus other biochemical markers, including hyaluronic acid [ 12 ], the Forns index [ 16 ], the APRI index [ 17 ] and the GlycoCirrhoTest [ 26 ]. All the comparisons were in favor of FT (Table 1 ) (Figure 1 , lower panel), except for the GlycoCirrhoTest, which has a similar AUROC (0.87 vs 0.89 for FT) [ 26 ]. Integrated database A total of 1,570 subjects were included in the integrated database. Of these, 1,270 were patients with chronic hepatitis C who tested PCR positive before treatment and who had had a liver biopsy and METAVIR staging and grading performed. Of these patients, 453 were from our center [ 11 , 14 ], including 130 patients coinfected with HCV and HIV [ 14 ]. Eight hundred and seventy (870) patients were from a multicentre study with a total of 398 patients assessed at inclusion and 419 at the end of follow-up six months after treatment; 352 being investigated twice. Three hundred (300) healthy blood donors were also included [ 20 ]. Diagnostic value of FT-AT according to HCV genotype and viral load There was no difference between the AUROC of FT-AT for the diagnosis of significant fibrosis (F2F3F4) (Figure 2A ) and significant necrosis (A2A3) (Figure 2B ) between 4 classes of genotype (1, 2, 3 and the rarer genotypes 4, 5, 6 grouped together). There was also no difference between the AUROC of FT-AT of patients with high or low viral loads for the diagnosis of significant fibrosis (Figure 2C ) or significant necrosis (Figure 2D ). Figure 2 Diagnostic values of FibroTest according to genotype and viral load. Graph A : AUROCs of FibroTest for the diagnosis of significant fibrosis, according to HCV genotypes. There were no significant differences: Genotype 1, n = 684, AUROC = 0.76, 95% Confidence Interval (95CI) = 0.72–0.79; genotype 2, n = 140, AUROC = 0.79, 95CI = 0.70–0.85; genotype 3, n = 143 AUROC = 0.76, 95CI = 0.67–0.83; other genotype, n = 46, AUROC = 0.72, 95CI = 0.52–0.85. Graph B : AUROCs of ActiTest for the diagnosis of significant necrosis, according to HCV genotypes. There were no significant differences: Genotype 1, n = 684, AUROC = 0.81, 95% Confidence Interval (95CI) = 0.77–0.84; genotype 2, n = 140, AUROC = 0.90, 95CI = 0.83–0.94; genotype 3, n = 143, AUROC = 0.79, 95CI = 0.71–0.85; other genotype, n = 46, AUROC = 0.76, 95CI = 0.57–0.87. Graph C : AUROCs of FibroTest for the diagnosis of significant fibrosis, according to serum HCV viral load. There were no significant differences: High viral load, n = 215, AUROC = 0.71, 95% Confidence Interval (95CI) = 0.64–0.78; Low viral load, n = 183, AUROC = 0.73, 95CI = 0.65–0.80. Graph D : AUROCs of ActiTest for the diagnosis of significant necrosis, according to serum HCV viral load. There were no significant differences: High viral load, n = 215, AUROC = 0.74, 95% Confidence Interval (95CI) = 0.64–0.82; Low viral load, n = 183, AUROC = 0.75, 95CI = 0.65–0.82. Diagnostic value of FT according to the independency of authors Among the 13 published studies of FT (detailed in Table 1 ), 9 studies estimated FT and 4 studies compared FT to other non-invasive tests. Among the 9 studies estimating FT, 5 were performed by the same single center (non-independent center), two were performed in totally independent centers, and two were performed in multiple centers, including the non-independent center. The AUROCs for the diagnosis of F2F3F4 versus random AUROCs at 0.50, were all significant and similar between these 3 groups in a meta-analysis: mean difference in AUROC = 0.29 (random effect model Chi-square = 549, P < 0.001), including 0.24 for independent, 0.25 for mixed and 0.36 for dependent studies. In the Callewaert et al. [ 26 ] study the AUROC of FT for the diagnosis of F4 was 0.89. Diagnostic value of FT-AT according to stage and grade The AUROCs between different stage combinations are given in Table 5 . Between two contiguous stages (one stage difference), the AUROCs were not significantly different and ranged from 0.63 to 0.71. Between patients with a two-stage difference, the AUROCs were not significantly different and ranged from 0.75 to 0.86. Between patients with a three-stage difference, the AUROCs were not significantly different and ranged from 0.87 to 0.95. Between patients with a four- or five-stage difference (blood donors versus F3 or F4, and F0 versus F4), the AUROCs were not significantly different and ranged from 0.95 to 0.99. Table 5 Summary of the diagnostic value of FibroTest for the diagnosis of all stage combinations of hepatic fibrosis, according to the AUROCs. F0 F1 F2 F3 F4 BD F0 F0F1 F1F2 F2F3F4 F3F4 Blood Donor (BD) n = 300 0.71 0.86 0.95 0.99 0.99 - 0.84 0.88 0.97 0.99 F0 n = 95 - F1 n = 688 0.66 - F2 n = 253 0.82 0.69 - F3 n = 111 0.92 0.80 0.63 - F4 n = 123 0.95 0.87 0.75 0.65 - BD F0 0.71 0.81 0.92 0.98 0.98 - F0F1 - - 0.71 0.82 0.88 - - - F1F2 0.71 - 0.69 0.81 0.82 0.84 - - - F2F3 0.85 0.76 - - 0.72 0.92 0.80 - - F3F4 0.94 0.81 0.81 - - 0.98 0.89 0.80 - F2F3F4 0.83 0.78 - - - 0.95 0.78 - - - BD F0F1 - - 0.77 0.87 0.91 - - - 0.83 0.89 The AUROC between all different stage combinations are given. Between two contiguous stages (one- stage difference), the AUROCs are given in bold. Between patients with a two-stages difference, the AUROCs are given in italics. Between patients with a three-stages difference, the AUROCs are given in bold and italics. Between patients with a four- or five-stages difference (blood donors versus F3 or F4, and F0 versus F4), the AUROCs are underlined. Significant differences were observed between AUROCs when there was a two-stage or more difference. The AUROCs between different grade combinations are given in Table 6 . Between two contiguous grades (one grade difference), the AUROCs were not significantly different and ranged from 0.60 to 0.70. Between patients with a two-grade difference, the AUROCs were not significantly different and ranged from 0.75 to 0.86. Between patients with a three-grade difference, the AUROCs were not significantly different and ranged from 0.87 to 0.95. Between patients with a four-grade difference (blood donors versus F3 and F0 versus F4), the AUROCs were not significantly different and ranged from 0.95 to 0.99. Table 6 Summary of the diagnostic value of ActiTest for the differential diagnosis of all grades of necroinflammatory hepatic activity, according to the AUROCs. A0 A1 A2 A3 BD A0 A0A1 A1A2 A2A3 Blood Donor BD n = 300 0.67 0.84 0.96 0.99 - 0.79 0.89 0.97 A0 n = 185 - A1 n = 443 0.69 - A2 n = 370 0.87 0.70 - A3 n = 272 0.93 0.79 0.60 - A0A1 - - 0.70 0.83 - A1A2 0.77 - - 0.70 0.85 - A2A3 0.89 0.74 - - 0.94 0.78 - A0A1A2 - - - 0.75 - - - - BD A0A1 - - 0.82 0.88 - - - 0.84 The AUROCs between all different grade combinations are given. Between two contiguous grades (one-grade difference), the AUROCs are given in bold. Between patients with a two-grades difference, the AUROCs are given in italics. Between patients with a three-grades difference, the AUROCs are given in bold and italics. Between patients with a four- or five-grades difference (blood donors versus F3 or F4, and F0 versus F4), the AUROCs are underlined. Significant differences were observed between AUROCs when there was a two-grade or more difference. Conversion between FT-AT results and the corresponding fibrosis stage and grade FT-AT is a continuous linear biochemical assessment of fibrosis stage and necroinflammatory activity grade. It provides a numerical quantitative estimate of liver fibrosis ranging from 0.00 to 1.00, corresponding to the well-established METAVIR scoring system of stages F0 to F4 and of grades A0 to A3. Among the 300 controls, the median FT value (± SE) was 0.08 ± 0.004 (95 th percentile, 0.23) and the median AT value was 0.07 ± 0.004 (95 th percentile, 0.26). Among the 1,270 HCV-infected patients, the FT conversion was 0.000 – 0.2100 for F0; 0.2101 – 0.2700 for F0–F1; 0.2701 – 0.3100 for F1; 0.3101 – 0.4800 for F1–F2; 0.4801 – 0.5800 for F2; 0.5801 – 0.7200 for F3; 0.7201 – 0.7400 for F3–F4; and 0.7401 – 1.00 for F4. (Figure 3A ). The AT conversion was 0.00 – 0.1700 for A0; 0.1701 – 0.2900 for A0–A1; 0.2901 – 0.3600 for A1; 0.3601 – 0.5200 for A1–A2; 0.5201 – 0.6000 for A2; 0.6001 – 0.6200 for A2–A3; and 0.6201 – 1.00 for A3 (Figure 3B ). The conversions are summarized in Figure 4 . Figure 3 Conversion between FibroTest and fibrosis stages, and between ActiTest and necroinflammatory activity grades – Graphs. Graph A : FibroTest values according to status, from blood donors to patients with cirrhosis (n = 1570). Graph B : ActiTest values according to status, from blood donors to patients with severe necrosis (n = 1570). F0 = no fibrosis, F1 = portal fibrosis, F2 = some septa, F3 = many septa, F4 = cirrhosis, A0 = no necroinflammatory activity, A1 = minimal activity, A2 = moderate activity, A3 = severe activity. (Consensus conferences recommend treatment in patients with either F2 stage or A2 grade.) Notched box plots showing the relationship between FibroTest and the stage of fibrosis (A) and between ActiTest and the grade of activity (B). The horizontal line inside each box represents the median, and the width of each box the median ± 1.57 interquartile range/√n (to assess the 95% level of significance between group medians). Failure of the shaded boxes to overlap signifies statistical significance (P < 0.05). The horizontal lines above and below each box encompass the interquartile range (from 25 th to 75 th percentile), and the vertical lines from the ends of the box encompass the adjacent values (upper: 75 th percentile plus 1.5 times interquartile range, lower 25 th percentile minus 1.5 times interquartile range). Figure 4 Conversion between FibroTest and fibrosis stages, and between ActiTest and necroinflammatory activity grades – Panels. Conversion between FibroTest and fibrosis stages using METAVIR, Knodell and Ishak fibrosis scoring systems (upper panel). Conversion between ActiTest and activity grades using METAVIR, Knodell and Ishak necroinflammatory activity scoring systems (lower panel). Discussion Based on the limitations of liver biopsy and the present overview of the diagnostic value of FT-AT, it seems that these non-invasive markers should be used as a first line assessment of liver injury in patients with chronic hepatitis C. Liver biopsy has three major limitations, which are the risk of adverse events [ 2 , 3 , 7 ], sampling error [ 4 - 6 ], and inter- and intra- pathologist variability [ 23 ]. An overview of published studies summarizes the risks of liver biopsy as pain (around 30%), severe adverse events (3/1,000) and death (3/10,000) [ 2 , 3 , 7 ]. Sampling variation is the major cause of variability [ 4 - 6 ]. In a study of patients with chronic hepatitis C that included only good quality biopsies, 30 of 124 patients (24.2%) had a difference of at least one grade, and 41 of 124 patients (33.1%) had a difference of at least one stage between the right and left lobes [ 4 ]. In 18 patients (14.5%), an interpretation of cirrhosis was made in one lobe, whereas stage 3 fibrosis was made in the other [ 4 ]. Recently, Bedossa et al. [ 6 ] observed very high coefficients of variation (55%) and high discordance rates (35%) for fibrosis staging in biopsies measuring 15 mm in length. The variability significantly improved in biopsies measuring 25 mm in length but was still very high with a 45% coefficient of variation and 25% discordance rate; the minimal variability was reached for biopsies, which were 40 mm in length [ 6 ]. Liver biopsy has also potential advantages. Biopsy could be of diagnostic value for other unrecognized liver disease. These events are probably rare in practice, as we observed no such a case in a prospective study of 537 consecutive patients with chronic hepatitis C [ 9 ]. For FT-AT it must be realized that the same predictive values were observed for patients coinfected with HIV [ 14 ], and in patients with other causes of liver fibrosis such as chronic hepatitis B [ 31 ], alcoholic liver disease [ 27 ] or non-alcoholic steato-hepatitis [ 27 ]. It is possible that biochemical markers such as those described here may provide a more accurate (quantitative and reproducible) picture of fibrogenic and necrotic events occurring within the liver than hepatic biopsy. The greater accuracies of FT-AT, when assessed with biopsy specimens greater than 15 mm versus smaller biopsies, suggest that some discordance between FT-AT and histology were due to biopsy specimen sampling error [ 8 ]. Several case reports have observed false negatives of liver biopsy versus biochemical markers [ 8 , 9 , 11 ]. The error was attributable to biopsy because there were overt clinical signs of cirrhosis such as esophageal varices, low platelet counts or a dysmorphic liver on ultrasound. In a recent prospective study we estimated that 18% of discordances between FT-AT and histology were attributable to biopsy failure (mostly due to small length) and 2% to FT-AT failure [ 9 ]. The present work allowed frequently asked questions to be answered, the first being whether the diagnostic values of FT-AT had been confirmed in all studies performed to date. A major strength of the studies pertaining to FT-AT is that they were carried out on a large number of patients with chronic hepatitis C, and the results were reproducible in different populations, including patients coinfected with HIV. There was a small variability in the AUROCs, both for the diagnosis of significant fibrosis (0.73 to 0.87) and significant necrosis (0.75 to 0.86). A weakness of this study was that the same group, which developed these tests, performed most of the published studies. However the independent published studies found the same significant diagnostic values than non-independent or multicentre studies. Several recent independent studies confirmed the predictive value of FT-AT [ 26 , 30 ]. The second question concerned the comparison of FT-AT to other tests. In their recent review, Gebo et al. [ 10 ] concluded that panels of markers might have the greatest value in predicting the absence or no more than minimal fibrosis on biopsy, and in predicting the presence of cirrhosis on biopsy (Evidence Grade B). They pointed out that five studies [ 11 , 32 - 35 ] used large panels of markers and achieved the greatest predictive values. Among these 5 studies were the first FT-AT study [ 11 ] and another study developed by the same group (combining age and platelets) [ 34 ]. A recent study compared FT-AT to the age and platelets index in the same patients and found that FT-AT was significantly better [ 15 ]. Three studies directly compared FT-AT, to hyaluronic acid [ 12 ], the Forns index [ 16 ] and the Wai index [ 17 ] in the same patients. FT-AT had higher diagnostic values (the AUROC was significantly higher). FT was in particular more sensitive for discriminating between F1 and F2, and more linearly correlated to stages when compared to those 3 other markers [ 12 , 16 , 17 ]. An additional weakness of the Forns index is the inclusion of cholesterol, which varies greatly in patients with genotype 3 [ 16 ]. The limitations of these three comparisons [ 12 , 16 , 17 ] are that they were retrospective and were performed by the same group. These comparisons, however, had no evident sources of bias. The comparison with the Forns Index [ 16 ] included all patients of the Imbert-Bismut et al. study (n = 323) [ 11 ], as the parameters belong to the routine biochemical tests. The comparison with the APRI index included 249/323 patients (77%) without any difference between included or non-included patients when all characteristics were compared [ 17 ]. The comparison with hyaluronic acid [ 12 ] included a total of 165 out of the 244 (68%) randomized patients pre-included. The 165 included patients did not differ from the 79 non-included patients according to the main characteristics. Among the 165 patients, the fibrosis index was assessed in 461 samples and hyaluronic acid in 457 samples [ 12 ]. Recently, a study using profiles of serum protein N-glycans found that a profile has a similar AUROC than FT for the diagnosis of compensated cirrhosis. When combined with FT this marker had 100% specificity and 75% sensitivity for the diagnosis of compensated cirrhosis, which is not significantly different from the 92% specificity and 67% sensitivity of the FT [ 26 ]. This study was independent and prospectively designed for taking FT as the comparison test. Only 24 patients with cirrhosis were included and no details were given concerning the causes of discordance between biopsy and biochemical markers. However FT-AT is the only panel of markers identified by an independent overview [ 9 ], which has been compared in the same patients with most of the other proposed markers. No studies were found that compared FT-AT with a panel of extra-cellular matrix markers [ 31 ]. Compared to other panels, FT-AT also allowed an estimation to be made not only of the fibrosis stage but also the necroinflammatory (histological) activity. The present analysis of the integrated database demonstrated that the diagnostic value of FT-AT did not depend on HCV genotype or viral load. However, because of the small number of patients included, studies in genotype 4, 5 and 6 would be useful. The present analysis also answered another frequently asked question concerning the predictive values for the intermediate stages of fibrosis. Contrary to the initial hypothesis, the diagnostic values of FT-AT for consecutive stages of fibrosis and grades of necroinflammatory activity were the same for both moderate and extreme stages and grades. Our interpretation is that the same overlap exists between all stages, which is mainly related to the sampling error of the biopsy. It is very reassuring that the medians of FT-AT are linearly associated with stages and grades (Figures 3A,3B ). The linearity of this association became even more evident as a larger number of patients were included (data not shown). Finally, the integrated database allowed a simple conversion system to be proposed to clinicians between liver injury as estimated by the FT-AT and that as estimated by liver biopsy (Figure 4 ). One conventional way to express the diagnostic values of FT-AT was summarized using the cutoffs of the distribution by stages and grades (Tables 2 and 4 ). The negative predictive value of FT for excluding significant fibrosis was excellent for the 0.31 cutoff (91%), as was the negative predictive value for excluding significant activity at the 0.36 cutoff of AT (85% negative predictive value). The positive predictive value of the 0.72 cutoff of FT for significant fibrosis was also high at 76%. This, however, may appear lower than the negative predictive value. There is a technical explanation owing to the prevalence of significant fibrosis, which was only 0.31 in this population. According to the excellent specificity (above 0.95), the positive predictive value increased rapidly in populations with more fibrosis (data not shown). We recently observed that the main reason for this was probably because most of the so-called false positives of the FT were in fact false negatives due to the small sampling size of liver biopsies [ 5 , 9 ]. The same comments can be made concerning the positive predictive value of AT for significant necrosis with 77% at the 0.60 cutoff. Again, it is probable that a large proportion of so-called false positives of AT were in fact false negatives due to liver biopsies which were too small. The ideal study would be one using biopsies measuring 40 mm in length, as two samples of 20 mm each during laparoscopy. Only this very high quality biopsy can be considered as a true gold standard. Obviously this type of biopsy cannot be performed routinely as first line, but it could be recommended for clinical research. Conclusions Based on these results, the use of the biochemical markers of liver fibrosis (FibroTest) and necrosis (ActiTest) can be recommended as an alternative to liver biopsy for the first line assessment of liver injury in patients with chronic hepatitis C. In clinical practice, liver biopsy should be recommended only as a second line test, i.e. , in case of high risk of error of biochemical tests or in transplanted patients. For clinical research, only very high quality liver biopsy (as two samples of 20 mm each) can be considered as a gold standard for validation of new alternatives. Methods Analysis of the literature We did a search for all publications and communications between February 2001 and March 2004 with the key words "FibroTest" and "ActiTest" in Medline and in the abstract books of hepatology, gastroenterology, internal medicine and infectious diseases annual meetings. Only publications or abstracts concerning FT-AT in chronic hepatitis C were included. Diagnostic value of FT-AT among published studies For each study we assessed the diagnostic value for the diagnosis of significant fibrosis (bridging fibrosis or stages F2, F3, F4 according to the METAVIR scoring system) and significant necroinflammatory activity (moderate or severe necrosis, grades A2 or A3 according to the METAVIR scoring system) by the area under the receiver operating characteristics curve (AUROC). For several databases it was possible to re-analyze the individual data and we looked at the sensitivity and specificity according to different thresholds (0.10, 0.30, 0.60 and 0.80). When FT-AT was compared to other biochemical tests, we also assessed the corresponding sensitivity and specificity according to several thresholds. Comparison of FT-AT diagnostic values with other biochemical markers We selected studies using direct comparisons of diagnostic values in the same patients. The AUROCs were compared for the diagnosis of significant fibrosis (F2F3F4) and significant necrosis (A2A3). Integrated database Patients were included in an integrated database if they belonged to a published population of patients with chronic hepatitis C. Liver biopsy was scored using the METAVIR scoring system and FT-AT was assessed using the recommended pre-analytical and analytical procedures [ 18 , 20 ]. A published population of 300 prospectively analyzed blood donors was included as a control group [ 20 ]. Diagnostic value of FT-AT according to HCV genotype and viral load Using the integrated database, we compared the AUROCs of FT-AT for the diagnosis of significant fibrosis (F2F3F4) and significant activity (A2A3) between 4 classes of genotype (1, 2, 3 and the rarer genotypes 4, 5, 6 grouped together). For viral load, only those assessed in the same laboratory were included in the comparison between AUROCs, and the median was used to define low and high viral loads (3,800,000 copies/ml) [ 8 ]. Diagnostic value of FT-AT according to stage and grade Using the integrated database, we compared the diagnostic values according to different stages or grades. We compared the AUROCs for all possible combinations of stages and grades, including combinations with blood donors. This allowed, for example, a comparison to be made of the diagnostic value of FT for discriminating between F1 and F2 after excluding all other stages of the database. Liver biopsies In the integrated database, liver biopsies were processed using standard techniques. A pathologist who was unaware of the biochemical markers evaluated fibrosis stage and necrosis grade according to the METAVIR scoring system [ 22 , 23 ]. Fibrosis was staged on a scale of 0 to 4: F0 = no fibrosis, F1 = portal fibrosis without septa, F2 = few septa, F3 = numerous septa without cirrhosis, F4 = cirrhosis. The grading of activity by the METAVIR system (based on the intensity of necroinflammatory activity, mainly on necrosis) was scored as follows: A0 = no necroinflammatory activity, A1 = mild activity, A2 = moderate activity, A3 = severe activity [ 22 , 23 ]. Biochemical markers We used the previously validated FT-AT [ 8 , 9 , 11 - 21 ]. FT-AT is a non-invasive blood test that combines the quantitative results of six serum biochemical markers [alpha2-macroglobulin, haptoglobin, gamma glutamyl transpeptidase (GGT), total bilirubin, apolipoprotein A1 and alanine aminotransferase (ALT)] with the patient's age and gender in a patented artificial intelligence algorithm (USPTO 6,631,330) to generate a measure of fibrosis stage and necroinflammatory grade in the liver. Statistical analysis Corresponding stages and grades were calculated from median scores and 95% confidence intervals were observed in 1,270 patients and 300 healthy blood donors. The AUROC was used as a measure of discrimination, estimated using the empirical (non-parametric) method by DeLong et al. [ 36 ], and were compared using the paired method by Zhou et al. [ 36 ]. All analyses are performed on the NCSS software (Kaysville, Utah) [ 36 ]. Authors' contributions TP and MM conceived the study, performed the statistical analysis, and wrote the manuscript. FIM, BH and DM carried out biochemical analyses. RP, DT, VR, and YB participated in the coordination of the study, and drafted the manuscript. AM participated in the design and coordination of assays in the control group. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC522750.xml
539303	Expression of inwardly rectifying potassium channels (GIRKs) and beta-adrenergic regulation of breast cancer cell lines	Background Previous research has indicated that at various organ sites there is a subset of adenocarcinomas that is regulated by beta-adrenergic and arachidonic acid-mediated signal transduction pathways. We wished to determine if this regulation exists in breast adenocarcinomas. Expression of mRNA that encodes a G-protein coupled inwardly rectifying potassium channel (GIRK1) has been shown in tissue samples from approximately 40% of primary human breast cancers. Previously, GIRK channels have been associated with beta-adrenergic signaling. Methods Breast cancer cell lines were screened for GIRK channels by RT-PCR. Cell cultures of breast cancer cells were treated with beta-adrenergic agonists and antagonists, and changes in gene expression were determined by both relative competitive and real time PCR. Potassium flux was determined by flow cytometry and cell signaling was determined by western blotting. Results Breast cancer cell lines MCF-7, MDA-MB-361 MDA-MB 453, and ZR-75-1 expressed mRNA for the GIRK1 channel, while MDA-MB-468 and MDA-MB-435S did not. GIRK4 was expressed in all six breast cancer cell lines, and GIRK2 was expressed in all but ZR-75-1 and MDA-MB-435. Exposure of MDA-MB-453 cells for 6 days to the beta-blocker propranolol (1 μM) increased the GIRK1 mRNA levels and decreased beta 2 -adrenergic mRNA levels, while treatment for 30 minutes daily for 7 days had no effect. Exposure to a beta-adrenergic agonist and antagonist for 24 hours had no effect on gene expression. The beta adrenergic agonist, formoterol hemifumarate, led to increases in K + flux into MDA-MB-453 cells, and this increase was inhibited by the GIRK channel inhibitor clozapine. The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a high affinity agonist for beta-adrenergic receptors stimulated activation of Erk 1/2 in MDA-MB-453 cells. Conclusions Our data suggests β-adrenergic receptors and GIRK channels may play a role in breast cancer.	Background Breast cancer is the leading cancer in women [ 1 ] and estrogen receptor (ER)(-) breast cancers have a poorer prognosis than ER(+) cancers [ 2 , 3 ]. Smoking is a controversial risk factor for the development of these malignancies [ 4 - 7 ]. However, increases in pulmonary metastatic disease and lung cancer have been seen in smokers with breast cancer [ 8 , 9 ]. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) causes cancer of the oral cavity, esophagus, respiratory tract and pancreas, but no breast cancer in laboratory animals [ 10 ] and has not been implicated in breast carcinogenesis to date. Recent studies in human cancer cell lines or in animal models have shown that the growth of adenocarcinomas of the lungs, pancreas and colon are under β-adrenergic control [ 11 - 15 ]. Studies in a cohort of 2442 men found an inverse association between risk of incident adenocarcinomas of the prostate and use of antihypertensive medication, including beta-blockers [ 16 ]. The tobacco-specific carcinogenic nitrosamine NNK has recently been identified as a high affinity β-adrenergic agonist that stimulated the growth of pulmonary and pancreatic adenocarcinomas in vitro and in animal models [ 11 , 13 , 15 ]. The expression of β-adrenergic receptors has been correlated with the over-expression of the arachidonic acid-metabolizing enzymes cyclooxygenase-2 (COX-2) and lipoxygenases (LOX) in adenocarcinomas of lungs [ 17 ], colon [ 18 ], prostate [ 19 ], and pancreas [ 15 ]. Inhibitors of these enzymes have been identified as cancer preventive agents in animal models of these cancers [ 13 , 20 - 22 ]. Collectively, these findings suggest that among the superfamily of adenocarcinomas at various organ sites, there is a subset of malignancies that is regulated by β-adrenergic and arachidonic acid-mediated signal transduction pathways. The majority of breast cancers are also adenocarcinomas and many of them over express COX-2 and/or LOX [ 23 ]. This raises the possibility that comparable to findings in adenocarcinomas of the lungs, pancreas, colon and prostate, a subset of breast cancers may also be under beta-adrenergic control. In support of this hypothesis, studies have demonstrated that three estrogen-responsive and three non-estrogen responsive human cell lines derived from breast adenocarcinomas demonstrated a significant reduction in DNA synthesis in response to beta-blockers or inhibitors of the arachidonic acid-metabolizing enzymes COX-2 and 5-LOX [ 24 ]. In addition, analysis by reverse transcription polymerase chain reaction (RT-PCR) revealed expression of β 2 -adrenergic receptors in all six breast cancer cell lines tested (MDA-MB-361, ZR-75-1, MCF-7, MDA-MB-453, MDA-MB-468, MDA-MB-435S), whereas β 1 receptors were not found in two estrogen non-responsive cell lines (MDA-MB-435S, MDA-MB-453) [ 24 ]. Expression of mRNA that encodes a G-protein coupled inwardly rectifying potassium channel (GIRK1) has been shown in tissue samples from approximately 40% of primary human breast cancers tested [ 25 ], and this expression of GIRK1 was associated with a more aggressive clinical behavior. Increases in GIRK currents by beta-adrenergic stimulation have been reported in adult rat cardiomyocytes and in Xenopus laevis oocytes coexpressing β 2 -adrenergic receptors and GIRK1/GIRK4 subunits [ 26 ]. In addition, in rat atrial myocytes transiently transfected with β 1 or β 2 adrenergic receptors, the beta-adrenergic agonist isoproterenol stimulated GIRK currents, whereas this stimulation was not seen in non-transfected cells [ 27 ]. The current investigations test the hypothesis that GIRK1 channels in human breast cancers are correlated with beta-adrenergic control. Methods Cell culture The ER(+) human breast cancer cell lines MDA-MB-361, ZR-75-1, and MCF-7 and the ER(-) cell lines MDA-MB-453, MDA-MB-468 and MDA-MB-435S were purchased from the American Type Culture Collection (Rockville, MD). Cells were maintained in RPMI 1640 medium supplemented with fetal bovine serum (10%, v/v), L-glutamine (2 mM), 100 U/ml of penicillin and 100 μg/ml streptomycin (Invitrogen-Life Technologies, Grand Island, NY) in an environment of 5% CO 2 . Exposure of cells to propranolol, isoproterenol, or clozapine (Sigma, St. Louis, MO), NNK (Chemsyn, Lexena, KS), or formoterol hemifumarate (Tocris, Ballwin, MO) for experiments was as detailed in the Figure Legends. RT-PCR RNA was isolated by Trizol reagent (Invitrogen-Life Technologies) or by an Absolutely RNA kit (Stratagene, La Jolla, CA). RT-PCR was done as previously described [ 28 ]. The GIRK1 primers are forward 5'-ctatggctaccgatacatcacag-3' and reverse 5'-ctgttcagtttgcatgcttcgc-3' which span exon 1 and 2 [ 29 ] and amplifies a 441 bp fragment (bases 631–1072, Genbank Acession # NM_002239). The GIRK2 primers are forward 5'-atggatcaggacgtcgaaag-3' and reverse 5'-atctgtgatgacccggtagc-3' amplifies a 438 bp fragment (bases 700–1137, Genbank Acession #U52153). The GIRK4 primers are forward 5'-aaccaggacatggagattgg-3' and reverse 5'-gagaacaggaaagcggacac-3' which amplifies a 401 bp fragment (bases 117–517, Genbank Acession # L47208). PCR conditions are 94°C, 30 sec; 55°C, 30 sec; 72°C, 45 sec for 40 cycles. Cyclophylin primers were used as an internal control (Ambion, Austin, TX). Relative competitive RT-PCR Preliminary experiments were done with MDA-MB-453 cells to determine a cycle number of PCR amplification that is within the linear range, which is critical for meaningful results to compare expression levels between samples and to determine the mixture of 18S primers/18S competimers (Ambion-Classic II). The 18S ribosomal RNA primers/competimers are used as an invariant internal control, which allows correction for sample variation. Results indicated this was 31 cycles of PCR and a 1:9 18S primer/competimers ratio. For experimental treatments, as described before [ 33 ], cDNA was made and PCR performed except reactions were spiked with 5 μCi [α- 32 P]-dCTP (3000 Ci/mmole, Dupont-NEN, Boston, MA). Reactions were run with the following conditions: 1 cycle of 2 min. at 94°C, then 31 cycles of 94°C, 30 sec; 55°C, 30 sec; 72°C, 45 sec. A 10 μl sample of each PCR reaction was heated at 95°C for 3 min., then loaded into a 5% TBE-urea Ready Gel (Bio-Rad, Hercules, CA). This underwent electrophoresis at 200 V in TBE buffer until the xylene cyanol dye front reached the bottom of the gel. The gel was transferred to filter paper, dried and exposed to film or imaged on a Molecular Dynamics 445 SI phosphoimager (Sunnyvale, CA). A 100 bp DNA ladder (Invitrogen-Life Technologies) was exchange labeled with T4 polynucleotide kinase and 30 μCi [γ- 32 P] ATP (3000 Ci/mM, Dupont-NEN). Real-time PCR The GIRK-1 primers for real time PCR are forward 5'-ctctcggacctcttcaccac-3' and reverse 5'-gccacggtgtaggtgagaat-3' (bases 398–477, Genbank Acession # NM002239). and the internal TaqMan probe is 6-FAM-tcaagtggcgctggaacctc-TAMRA (bases 429–449, Sigma-Genosys, The Woodlands, TX), annealing temperature 62°. GIRK2 primers-forward 5'-gacctgccaagacacatcag-3' and reverse 5'-cggtcaggtagcgataggtc-3' (bases 766–886, Genbank Acession # U52153) and the internal TaqMan probe is 6-FAM-gtgcaatgttcatcacggcaac-TAMRA (bases 837–859), annealing temperature 56°. GIRK4 primers-forward 5'-agcgctacatggagaagagc-3' and reverse 5'-aagttgaagcgccacttgag-3' (bases 241–358, Genbank Acession # L47208) and the internal TaqMan probe is 6-FAM-accggtacctgagtgacctcttca-TAMRA (bases 301–324), annealing temperature 62°. Reactions were run on a Cepheid SmartCycler (Sunnyvale, CA). Reaction conditions are 200 μM dNTPs, 0.3 μM gene specific primers, 0.2 μM TaqMan probe, 4 mM (GIRK1) or 6 mM (GIRK2or4) magnesium acetate, 2 μl cDNA and 1.5 U MasterTaq (Eppendorf, Westbury, NY) and MasterTaq buffer in a final volume of 25 μl. TaqMan beta-actin detection reagents (Applied Biosystems) were used with the same reaction conditions as above except a 5 mM magnesium concentration was used and this was run at 95° for 120 seconds, followed by 45 cycles of 95°, 15 seconds; 68°, 30 seconds. Measurement of potassium flux We determined inward potassium flux in these cells by flow cytometry via the method of Krjukova et al. [ 30 ]. The negatively charged fluorescent dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBaC 4 (3)) (Molecular Probes, Eugene, OR) was added to MDA-MB-453 breast cancer cell line suspensions of 1 × 10 6 cells at a final concentration of 150 × 10 -9 M. Fluorescence intensity measurement after treatment of the cells was obtained from a FACS Vantage/SE Cell Sorter (San Jose, CA). Analysis of protein expression by western blots Following incubation with agents as detailed in the Figure legends, cells were washed twice with phosphate buffered saline and lysed with cold RIPA lysis buffer containing protease inhibitors (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 1% Triton × 100, 0.1% SDS, 1% sodium deoxycholate, 1 mM EDTA, 50 mM NaF, 10 mM sodium pyrophosphate, 0.5 mM DTT). Cell lysates were collected from culture plates using a rubber policeman, and protein collected by centrifugation. Protein concentrations were determined by BCA protein assay (Pierce, Rockford, IL). Aliquots of 20 μg protein were boiled in 2x loading buffer (0.1 M Tris-Cl, pH 6.8, 4% SDS, 0.2% Bromophenyl blue, 20% glycerol) for 4 minutes, then loaded onto 10% Tris-HCl-Polyacrylamide gels (Biorad, Hercules, CA), and transferred electrophoretically to nictrocellulose membranes. Membranes were incubated with primary antibodies (phospho-Erk; Cell Signaling, Beverly, MA) and appropriate secondary antibodies (Cell Signaling or Rockland, Gilbertsville, PA or Molecular Probes, Eugene OR). In all western blots, membranes were additionally probed with an antibody for actin (Sigma) to ensure equal loading of protein between samples. The antibody-protein complexes were detected as previous described [ 28 ] or by the LiCor Odyssey infrared imaging system (Lincoln, NE). Results The estrogen-responsive (MCF-7, ZR-75-1, MDA-MB-361) and estrogen non-responsive (MDA-MB-453, MDA-MB-435S, MDA-MB-468) human breast cancer cell lines were screened for the presence of the GIRK1 potassium channel by RT-PCR analysis. The ER(+) cell lines MCF-7, MDA-MB-361 and ZR-75-1 and the ER(-) cell line MDA-MB-453 expressed mRNA for the GIRK1 channel (Figure 1 ). The ER(-) cell lines MDA-MB-468 and MDA-MB-435S did not express GIRK1 (Figure 1 ). GIRK1 is also not expressed in the normal breast epithelial cell line MCF 10A (data not shown). The PCR product from the MDA-MB-453 cell line was sequenced to verify the integrity of the PCR process and found to be homologous to the published sequence (data not shown). The PCR primers were designed to span exon 1 and 2 of GIRK1 [ 29 ]. In addition PCR amplification of negative control reactions (without the reverse transcriptase enzyme, data not shown) indicated that this was actually representative of mRNA expression and there was no contaminating genomic DNA. Since the GIRK1 potassium channels work as heterotetramers, we needed to determine which other GIRK channels were expressed in these breast cancer cell lines. As determined by RT-PCR, GIRK4 was expressed in all six breast cancer cell lines (Figure 2 ), and GIRK2 was expressed in four of the six cell lines. GIRK2 was not expressed in ZR-75-1 or MDA-MB-435S cell lines (Figure 2 ). To determine if GIRK channels are functionally linked with β-adrenergic receptors in breast cancer cells expressing this ion channel, we decided to investigate the ER(-) cell line MDA-MB-453. This ER (-) cell line, which was the only ER(-) cell line tested that expressed GIRK1, was used for further experiments due to the fact that ER(-) breast cancers have a poorer prognosis than ER(+) cancers [ 2 , 3 ]. In addition, previous research in our laboratories indicated that this cell line expressed the β 2 adrenergic receptor but not the β 1 receptor [ 24 ]. MDA-MB-453 were continuously exposed to the beta-blocker propranolol (1 μM) for 6 days. Previous results from our laboratories indicated that maximal inhibition of breast cancer cell proliferation was at 1 μM propranolol [ 24 ]. Using relative RT-PCR, we saw a significant increase in GIRK1 channel mRNA expression (1.6 fold, Figure 3 ) after 6 days of continuous exposure to propranolol (p < 0.0001 by t-test). In these experiments, propranolol was added fresh each day. We also saw a significant decrease (1.5 fold) in β 2 -adrenergic receptor mRNA (p < 0.0079 by t-test) (data not shown). Using the same cDNA samples, we performed a real-time RT-PCR assay using GIRK1 primers designed for real-time PCR and a TaqMan probe. We also saw a significant increase of GIRK1 mRNA using this method (Figure 4 ) and no change in the control, beta-actin values. Threshold values (C T ) were calculated for each sample, which will be lower for samples with more mRNA expression. C T values for GIRK 1 expression were significantly (p < 0.001 by t-test) lower for propranolol treated cells (27.808 ± 0.107) (SD) as compared to control MDA-MB-453 cells (28.964 ± 0.338) (SD). Actin C T values were unchanged between control (13.666 ± 0.286) (SD) and propranolol treated cells (13.404 ± 0.427) (SD). The exposure to propranolol caused a slight decrease in GIRK2 mRNA expression (p < 0.04 by t-test) in the treated cells, opposite the result we found for GIRK1. Control (C T cycle values-31.35 ± 0.73) and propranolol (C T cycle values-32.24 ± 0.38). GIRK4 expression levels were unchanged between control and propranolol treated cells, indicated by real time PCR. Control (C T cycle values-33.0 ± 2.3) and propranolol treated (C T cycle values-31.7 ± 0.38). By contrast, MDA-MB-453 cells treated for 30 minutes daily for 7 days with 1 μM propranolol did not show changes in GIRK1 mRNA expression levels (Figure 5 ). No significant differences in GIRK1 mRNA expression were seen when MDA-MB-453 cells were exposed to 1 μM of either propranolol or the broad spectrum β-adrenergic agonist isoproterenol for 24 hours (data not shown). Although the gene expression studies showed no effects at shorter time periods or when it was not in the media constantly, we wished to determine if other cellular function are affected at shorter time periods in MDA-MB-453 cells. Potassium flux into cells would be an important part of any cellular response involving GIRK channels. We determined inward potassium flux in MDA-MB-453 cells by flow cytometry. The negatively charged fluorescent dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBaC 4 (3)) was added to MDA-MB-453 breast cancer cell line suspensions of 1 × 10 6 cells at a final concentration of 150 × 10 -9 M. Fluorescence intensity measurement after treatment of the cells was obtained from a FACS Vantage/SE Cell Sorter. An increase of dye fluorescence corresponds to membrane potential depolarization and K + flux. The β 2 selective agonist, formoterol hemifumarate (1 μM), added to MDA-MB-453 cell suspensions at the same time as the fluorescent dye lead to a 2X increase of fluorescence inside the cells, indicating inward potassium movement (Figure 6A & 6B ). Dye alone added to cells had no effect (data not shown). The GIRK inhibitor clozapine [ 31 ] (50 μM) added just prior to dye and formoterol addition completely blocked the effect of the beta-adrenergic agonist formoterol, (Figure 6C ) indicating that blockage of the GIRK channel inhibited potassium flux, and that effects of beta-adrenergic agents on this breast cancer cell line are indeed mediated by GIRK channels. We also determined signaling events in MDA-MB-453 cells that are affected by beta-adrenergic agents. Increased activation of Erk 1/2 was seen in MDA-MB-453 cells after treatment with 100 pM NNK at times ranging from 15–150 minutes (Figure 7 ). The concentration of NNK used by us is within the range of systemic NNK concentrations found in smokers. In addition, an experiment in Patas monkeys [ 32 ] has shown blood levels of 1.6 pg/ml (7.72 × 10 -12 M) after exposure to a dose of tritiated NNK equivalent to the amount of NNK found in two packs of cigarettes. Stimulation of Erk 1/2 was also seen using 1 μM of the beta-adrenergic agonist formoterol, but only at 150 minutes (data not shown). Discussion Our data demonstrate expression of the G-protein inwardly rectifying potassium channel 1 (GIRK1) in 67% of the breast cancer cell lines tested, with higher levels in ER(+) cell lines. Approximately 40% of primary human breast cancers were found to express GIRK1 and expression of GIRK1 was not found to be correlated with ER status [ 25 ]. These differences in our studies may be due to the subset of breast cancer cell lines tested. We also found that the normal breast epithelial cell line MCF 10A lacked GIRK1 expression (data not shown). GIRK1 cannot form functional channels by itself, other GIRK channels are needed [ 33 ]. All six breast cancer cell lines tested express either GIRK2 or GIRK4 indicating that functional GIRK potassium channels are possible in these breast cancer cell lines. The majority of experiments in the present study were done with the ER(-) cell line MDA-MB-453 since it was the only ER(-) cell line tested that expressed GIRK1, and because ER(-) breast cancers have a poorer prognosis than ER(+) cancers [ 2 , 3 ]. We saw a significant increase in GIRK1 channel mRNA expression after 6 days of continuous exposure to propranolol in MDA-MB-453 cells. It is clear that at least six days of continuous exposure to the beta-blocker propranolol is necessary to effect gene expression. Gene expression of β 2 -adrenergic mRNA was decreased by the same treatment (data not shown). Addition of propranolol for 7 days for only 30 minutes daily had no effect on GIRK1 gene expression. Treatment for a shorter period of time (24 hours) also had no effect on GIRK1gene expression in our studies. The 6 day continuous exposure to propranolol caused a barely detectable decrease in GIRK2 mRNA expression and no change in GIRK4 mRNA expression levels. Longer treatment times may be necessary for gene expression changes in GIRK2 or GIRK4 similar to gene expression changes that are seen in GIRK1. Although there were no short-term effects of beta-adrenergic agents on GIRK gene expression, we detected other cellular effects. The beta-adrenergic agonist formoterol hemifumarate stimulated potassium influx in MDA-MB-453 cells, and this influx was prevented by the GIRK channel inhibitor clozapine. NNK, a high affinity agonist for beta-adrenergic receptors [ 11 ] increased activation of Erk 1/2 in MDA-MB-453 breast cancer cells. Formoterol also increased activation of Erk 1/2, but to a lesser degree (data not shown). Previous studies indicated that the beta-adrenergic agonist isoproterenol stimulates growth [ 24 ]. GIRK currents have been shown to be increased in cells stimulated with the beta-adrenergic agonist isoproterenol in rat atrial myocytes transfected with β 1 or β 2 receptors [ 27 ]. Heterologous facilitation of GIRK currents by β-adrenergic stimulation was also seen in rat cardiomyocytes [ 26 ]. Two polymorphisms in the β 2 and β 3 adrenergic receptors were found to be correlated with a decreased risk for breast cancer [ 34 ], suggesting an important role of this receptor family in the genesis of breast cancer. In previous work, we demonstrated mRNA expression by RT-PCR of the β 2 adrenergic receptor in the six breast cancer cell lines used in this study, but expression of β 1 in all the estrogen responsive cell lines but not in two ER(-) cell lines (MDA-MB-435S and MDA-MB-453) [ 24 ]. Further studies are needed to determine how GIRK1(+) and ER(-) breast cancers are regulated and if GIRK channel agonists and antagonists have effect on proliferation in breast cancer. It also remains to be determined if this same regulation is present in GIRK1(+) and ER(+) breast cancer malignancies. This is of particular importance since a recent report indicated that 17-β-estradiol can modulate GIRK channel activation in the brain [ 35 ]. Future studies are also needed to determine if GIRK3 is involved in breast cancer. However, we think this unlikely because one of the functions of GIRK3 is to inhibit plasma membrane expression of other GIRK subunits [ 36 ]. Conclusions All six breast cancer cell lines tested express either GIRK2 or GIRK4 indicating that functional GIRK potassium channels are possible in these breast cancer cell lines. This is the first report that implicates β-adrenergic receptors and G-protein inwardly rectifying potassium channels 1 (GIRK1) in the regulation of human breast cancer cells and suggests a potential role of the tobacco nitrosamine NNK in breast cancers expressing these regulatory pathways. Beta-adrenergic antagonists have both long term effects on gene expression and beta-adrenergic agonists have short term effect on potassium flux and cellular signaling pathways. Competing interests The author(s) declare they have no competing interests. Authors' contributions HP carried out the majority of experiments and participated in the design of the study, and helped draft the manuscript. QY carried out the western blots. YC was involved in relative competitive RT-PCR studies. HS conceived of the study and helped draft the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC539303.xml
533885	Punica granatum (Pomegranate) juice provides an HIV-1 entry inhibitor and candidate topical microbicide	Background For ≈ 24 years the AIDS pandemic has claimed ≈ 30 million lives, causing ≈ 14,000 new HIV-1 infections daily worldwide in 2003. About 80% of infections occur by heterosexual transmission. In the absence of vaccines, topical microbicides, expected to block virus transmission, offer hope for controlling the pandemic. Antiretroviral chemotherapeutics have decreased AIDS mortality in industrialized countries, but only minimally in developing countries. To prevent an analogous dichotomy, microbicides should be: acceptable; accessible; affordable; and accelerative in transition from development to marketing. Already marketed pharmaceutical excipients or foods, with established safety records and adequate anti-HIV-1 activity, may provide this option. Methods Fruit juices were screened for inhibitory activity against HIV-1 IIIB using CD4 and CXCR4 as cell receptors. The best juice was tested for inhibition of: (1) infection by HIV-1 BaL, utilizing CCR5 as the cellular coreceptor; and (2) binding of gp120 IIIB and gp120 BaL, respectively, to CXCR4 and CCR5. To remove most colored juice components, the adsorption of the effective ingredient(s) to dispersible excipients and other foods was investigated. A selected complex was assayed for inhibition of infection by primary HIV-1 isolates. Results HIV-1 entry inhibitors from pomegranate juice adsorb onto corn starch. The resulting complex blocks virus binding to CD4 and CXCR4/CCR5 and inhibits infection by primary virus clades A to G and group O. Conclusion These results suggest the possibility of producing an anti-HIV-1 microbicide from inexpensive, widely available sources, whose safety has been established throughout centuries, provided that its quality is adequately standardized and monitored.	Background The global AIDS epidemic has proceeded relentlessly for ≈ 24 years with no promising prophylactic intervention in sight. In 2003 there were 5 million new HIV infections, and 3 million AIDS deaths [ 1 ]. To date the number of individuals living with HIV-1 infection/AIDS has reached 40 million, and ≈ 30 million people have already died from AIDS since the beginning of the pandemic [ 1 , 2 ]. Most new infections have been acquired by the mucosal route, heterosexual transmission playing the major (≈ 80%) role. Although the incidence of transmission per unprotected coital act is estimated to be low (0.0001 – 0.004), but strikingly increased when acutely infected individuals are involved [ 3 , 4 ], the cumulative effect is overwhelming. Anti-HIV-1 vaccines applicable to global immunization programs are not expected to become available for many years. Thus, other prevention strategies are urgently needed. This includes educational efforts and application of mechanical and/or chemical barrier methods. The latter correspond to microbicides, i.e. topical formulations designed to block HIV-1 infection (and possibly transmission of other sexually transmitted diseases) when applied vaginally (and possibly rectally) before intercourse [ 3 , 5 - 7 ]. Conceptually, it is preferred that the active ingredient(s) of microbicide formulations (1) block virus entry into susceptible cells by preventing HIV-1 binding to the cellular receptor CD4, the coreceptors CXCR4/CCR5 and to receptors on dendritic/migratory cells (capturing and transmitting virus to cells which are directly involved in virus replication), respectively [ 3 , 8 - 11 ], and/or (2) are virucidal. The formulations must not adversely affect the target tissues, and should not cause them to become more susceptible to infection after microbicide removal [ 12 , 13 ]. Treatment with anti-retroviral drugs has decreased mortality from AIDS in industrialized countries but has had a minimal effect so far in developing countries [ 14 ]. To avoid a similar dichotomy with respect to microbicides, they should be designed and selected to become affordable and widely accessible, while shortening the time between research and development and their marketing and distribution as much as possible. This would be facilitated if mass manufactured products with established safety records were to be found to have anti-HIV-1 activity. Qualifying candidates to be considered for microbicide development may possibly be discovered by screening pharmaceutical excipients (= "inactive" ingredients of pharmaceutical dosage forms) and foods, respectively, for anti-viral properties. This approach has already led to the discovery of cellulose acetate 1,2-benzenedicarboxylate (used for coating of enteric tablets and capsules) as a promising candidate microbicide [ 15 - 19 ]. Here we report the outcome of screening fruit juices neutralized to pH ≈ 7 to discount nonspecific effects caused by acidity. Methods Reagents Pomegranate juices (PJ) were purchased in local New York City stores; their origin is given in parentheses: PJ1 (Madeira Enterprises Inc., Madeira, CA); PJ2 was prepared from fresh ripe pomegranates in our laboratory; PJ3 (Sadaf ® ; Sadaf ® Foods, Los Angeles, CA; additional ingredients: fructose, citric acid); PJ4 (Cortas Canning & Refrigeration Co. S.A.L., Beirut, Lebanon); PJ5 (Kradjian, Import & Wholesale Distribution, Glendale, CA. Product of Iran); PJ6 (R.W. Knudsen ; Just Pomegranate; Knudsen & Sons, Inc., Chico, CA); PJ7 (Aromaproduct Ltd., Product of Georgia; distributed by Tamani, Inc., New York, NY). Starches used were: PURE-DENT ® B815 Corn Starch NF, PURE-DENT ® B816 Corn Starch USP, Spress ® B825 Pregelatinized corn starch NF, Spress ® B820 Pregelatinized corn starch NF, INSTANT PURE-COTE™ B792 Food starch-modified, INSCOSITY™ B656 Food starch-modified (Grain Processing Corporation, Muscatine, IN); PURITY ® 21 corn starch NF and PURITY ® 826 corn starch NF (National Starch and Chemical Company, Bridgewater, NJ); Remyline AX-DR Waxy rice starch and Remy DR native rice starch, medium grind (A&B Ingredients, Fairfield, NJ); ARGO ® corn starch (Best Foods Division, CPC International Inc., Engelwood Cliffs, NJ); STALEY ® pure food powder starch (Tate & Lyle, Decatur, IL); STARCH 1500 pregelatinized starch NF (Colorcon, West Point, PA). The following polymers were used: polyethylene glycols (PEG) 1000 NF, 1500 NF and 8000 NF; and hydroxypropyl methylcellulose, 50 cps, USP (Spectrum, New Brunswick, NJ); Carbopol 974P-NF (B. F. Goodrich Co., Cleveland, OH); Carbophil, Noveon AA1 (Noveon, Inc., Cleveland OH); and Pharmaburst B2 (SPI Pharma, New Castle, DE). Fattibase was from Paddock Laboratories, Inc., Minneapolis, MN. Recombinant proteins employed were: HIV-1 IIIB gp120, biotinyl-HIV-1 IIIB gp120, CD4, and biotinyl-CD4 (ImmunoDiagnostics, Inc., Woburn, MA); HIV-1 IIIB BaL gp120 and FLSC (a full length single chain protein consisting of BaL gp120 linked with the D1D2 domains of CD4 by a 20 amino acid linker) (produced in transfected 293T cells [ 20 ]). Phycoerythrin (PE)-labeled streptavidin was from R & D Systems, Minneapolis, MN. Biotinylated Galanthus nivalis lectin was from EY Laboratories, Inc. San Mateo, CA. Rabbit antibodies to synthetic peptides from gp120 (residue numbering as in reference [ 21 ]) were prepared as described [ 21 ]. Monoclonal antibodies (mAb) 588D, specific for the CD4 binding site, and 9284, specific for the gp120 V3 loop, were from Dr. S. Zolla-Pazner and NEN Research Products, Du Pont, Boston, MA, respectively. A "generic" version of the nonnucleoside HIV-1 reverse transcriptase inhibitor TMC-120 [ 22 ] was synthesized by Albany Molecular Research, Inc., Albany, NY, and used in control experiments at a final 5 μM concentration. Pelletted, 1000-fold concentrates of HIV-1 IIIB (6.8 × 10 10 virus particles/ml) and BaL (2.47 × 10 10 virus particles/ml) were from Advanced Biotechnologies, Inc., Columbia, MD. Primary HIV-1 isolates, MT-2 cells, HeLa-CD4-LTR-β-gal and U373-MAGI-CCR5E cells (both contributed by Dr. Michael Emerman) and Cf2Th/synCCR5 cells (contributed by Dr. Tajib Mirzabekov and Dr. Joseph Sodroski) were obtained from the AIDS Research and Reference Reagent Program operated by McKesson BioServices Corporation, Rockville, MD. CEMx174 5.25M7 cells, transduced with an HIV-1 long terminal repeat (LTR)-green fluorescent protein and luciferase reporter construct, expressing CD4 and CXCR4 and CCR5 coreceptors [ 23 ], were obtained from Dr. Cecilia Cheng-Mayer. The cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1 μg/ml puromycin and 200 μg/ml G418. These cells are suitable for titration of both X4 and R5 HIV-1 isolates and for determining the effectiveness of anti-HIV-1 drugs with reliable reproducibility. This is impossible to accomplish by using peripheral blood mononuclear cells (PBMCs) because of their variations in susceptibility to HIV-1 infection among cells derived from distinct individuals [ 24 - 26 ]. PBMCs were isolated from HIV-1 negative donors as described [ 27 ]. Formulations In attempts to separate gp120-CD4 binding inhibitory activity from most other ingredients of PJ, 200 mg of distinct starch preparations were added per ml of PJ1. After mixing for 1 h at 20°C, excess juice was decanted, and the pellets resuspended in 1 ml of distilled water. Based on results of enzyme linked immunosorbent assays (ELISA), PURITY ® 21 corn starch, NF grade (S21) was selected for further studies, and the corresponding PJ complex was designated as PJ-S21. PJ-S21 was freeze dried and used to prepare the following formulations: PEG suppositories (50% PJ-S21, 45% PEG 1000, 5% PEG 1500); Fattibase suppositories (50% PJ-S21, 50% Fattibase); and mucoadhesive instantly dispersible tablets (50% PJ-S21, 20% HPMC, 20% Pharmaburst, 7.5% Carbopol 974P and 2.5% Carbophil). Enzyme linked immunosorbent assays (ELISA) Inhibition of infection by HIV-1 IIIB and BaL, respectively, was determined relying on a β-galactosidase readout system [ 18 ]. The enzyme was quantitated with a Galacto-Light Plus System chemiluminescence reporter assay (Applied Biosystems, Foster City, CA) using a Microlight ML 2250 luminometer (Dynatech Laboratories, Inc., Chantilly, VA). To measure virucidal activity, virus was separated from excess inactivating agent by centrifugation and/or precipitation with PEG 8000 [ 18 , 19 ]. Serial dilutions of the treated virus were assayed for infectivity as described above. Dose response curves (i.e. luminescence vs. dilution) for treated and control viruses were obtained, and the percentages of virus inactivation were calculated [ 19 ]. To determine inhibition of infection by primary HIV-1 strains, CEMx174 5.25 M7 cells were incubated with 100 × TCID 50 of primary HIV-1 strains in the absence or presence of PJ-S21 at graded concentrations for 3 days at 37°C. The experiments were done in triplicate. Infection was quantitated by measuring luciferase activity [ 23 ] using a kit from Promega (Madison, WI) in an Ultra 384 luminometer (Tecan, Research Triangle Park, NC). CD4-HIV-1 gp120 binding and its inhibition were measured by ELISA. Wells of 96-well polystyrene plates (Immulon II, Dynatech Laboratories, Inc., Chantilly, VA) were coated with 100 ng/well of either gp120 IIIB or gp120 BaL, and post-coated as described [ 16 ]. Dilutions of PJs and of PJ-S21, respectively, in 0.14 M NaCl, 0.01 M Tris, 0.02% sodium merthiolate, pH 7.0 (TS) containing 100 μg/ml bovine serum albumin (BSA) were added to the wells for 1 h at 37°C. The wells were washed 5 × with TS. Biotinyl-CD4 (1 μg/ml) in TS-1% gelatin was added to the wells for 5 h at 37°C. After washing 1 × with TS-0.1% Tween 20 and 5 × with TS, horseradish peroxidase (HRP)-streptavidin (0.625 μg/ml; Amersham, Arlington Heights, IL) in TS-2% gelatin-0.05% Tween 20 was added. After 30 min at 37°C, the wells were washed 4 × with TS-0.1% Tween 20 and 2 × with TS. Bound HRP was detected using a kit from Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD) and the absorbance (A) read at 450 nm. A in the absence of inhibitors was 1.0 to 1.5, and 0 to 0.005 in the absence of biotinyl-CD4. In an alternative assay, CD4 (500 ng/ml) was mixed with biotinyl-gp120 (1 μg/ml) in the presence or absence of inhibitors for 30 min at 20°C. Serial dilutions of the mixtures were added to wells coated with the anti-CD4 mAb OKT 4 (Ortho-Clinical Diagnostics, Rochester, NY) and captured biotinyl-gp120 was detected with HRP-streptavidin as described above. To measure binding to gp120 of antibodies to gp120 peptides, the respective rabbit antisera were diluted 50-fold in a mixture of FBS and goat serum (9:1) containing 0.1% Tween 20 (pH 8.0) and added to gp120 wells. Bound IgG was detected with HRP labeled anti-rabbit IgG (Sigma, St. Louis, MO; 1 μg/ml in TS-10% goat serum-0.1% Tween 20). A cell-based ELISA was used to measure the blocking of CCR5 binding sites on HIV-1 BaL gp120 by PJ and PJ-S21, respectively [ 20 ]. Briefly, FLSC (125 ng/ml) in the absence or presence of graded amounts of inhibitors was added to Cf2Th/synCCR5 cells fixed with 5% formaldehyde in wells of 96-well plates. After 1 h at 37°C, bound FLSC was detected with mAb M-T441 (125 ng/ml; Ancell, Bayport, MN) specific for the CD4 D2 domain, followed sequentially by biotinylated anti-mouse IgG and HRP-streptavidin. Results Anti-HIV-1 activity of pomegranate juice Serial twofold dilutions of juices [apple, black cherry, blueberry, coconut milk, cranberry, elderberry, grape (red), grapefruit, honey, lemon, lime, pineapple, pomegranate and red beet (10% reconstituted dry powder)] were assayed for inhibition of infection by HIV-1 IIIB of cells expressing the CD4 and CXCR4 receptors and coreceptors. Most juices (4-fold diluted) had no inhibitory activity, except blueberry, cranberry, grape and lime juice, respectively [endpoints for 50% inhibition of infection (ED 50 ) between 1/16 and 1/64]. Consistently, PJs from distinct geographical areas had the highest inhibitory activity (Fig. 1 ; blue shaded area). Since HIV-1 viruses utilizing CCR5 as coreceptor (=R5 viruses) are predominantly transmitted sexually [ 3 , 28 ], it was important to test whether PJ can inhibit not only infection by HIV-1 IIIB, a virus utilizing CXCR4 as coreceptor (=X4 virus), but also infection by an R5 virus, HIV-1 BaL. Results in Fig. 1 (red shaded area) show that infection by the latter virus is also inhibited, albeit less effectively than that by HIV-1 IIIB. Blocking virus entry is a primary target for microbicide development [ 3 , 8 - 11 ]. Therefore, it was of interest to determine whether or not PJ inhibited the binding of the HIV-1 envelope glycoprotein gp120 to CD4, the common receptor for both X4 and R5 viruses. Pretreatment of both gp120 IIIB and BaL by PJ inhibited subsequent binding of soluble labeled CD4 (Fig. 2 ). This suggested that one or more PJ ingredients bound strongly or irreversibly to the CD4 binding site on gp120. These results, obtained in an ELISA using gp120 immobilized on polystyrene plates, were confirmed in another assay in which both gp120 and CD4 were in soluble form (data not shown). In reverse experiments, pretreatment of CD4 with PJ failed to block subsequent gp120 binding. Other juices having anti-HIV-1 activity (blueberry, cranberry, grape and lime) failed to block gp120-CD4 binding. To delineate sites on gp120 blocked by the PJ inhibitor(s), the inhibitory effect of PJ on binding to gp120 IIIB of antibodies to peptides derived from the amino acid sequence of gp120 was studied. The binding of antibodies to peptides (102–126), (303–338), (306–338), (361–392), (386–417), (391–425), (411–445) and (477–508) was significantly (≥ 50%) inhibited (Fig. 3 ). The binding to gp120 IIIB of monoclonal antibodies 9284 and 588D, specific for the gp120 V3 loop (residues 303 – 338) and the CD4 binding site, respectively [ 29 , 30 ] was each inhibited by 97%. Some of the relevant peptides contain residues involved in CD4 binding [ 31 - 33 ] while all discerned peptides include residues involved in coreceptor binding [ 34 - 39 ]. The locations of the peptides and of residues involved in receptor/coreceptor binding on the X-ray crystallographic structure of gp120 are shown in Fig. 4 . These results suggest that the PJ inhibitor(s) may also block gp120-coreceptor binding. This will be addressed subsequently. Separation of anti-HIV-1 inhibitor(s) from pomegranate juice PJ is intensely colored. Therefore, it cannot be directly formulated into a microbicide since it would stain clothing, which is unacceptable. Attempts were made to separate or isolate the active ingredient(s) from PJ. After striving intermittently for over four years to accomplish this, it was discovered that the inhibitor(s) of gp120-CD4 binding can be adsorbed effectively (≥ 99%) onto a selected brand of corn starch (Fig. 5 ), resulting in a nearly colorless product, designated as PJ-S21. PJ-S21, suspended in water or unbuffered 0.14 M NaCl had a pH of 3.2, compatible with the acidic vaginal environment in which it would remain stabile after application (see below). Inhibitors of gp120-CD4 binding could be eluted from PJ-S21 by extraction with ethanol/acetone 6:4. Drying of the extract followed by gravimetry indicated that the extract contained 3.17 mg solids per gram of PJ-S21. PJ-S21, to the same extent as the original PJ, inhibited the binding of gp120 IIIB-CD4 complexes to cells expressing CXCR4, as determined by flow cytometry (Fig. 6 ). Similarly, binding of a gp120 BaL-CD4 fusion protein to cells expressing CCR5 was blocked by PJ and PJ-S21, as detemined by a cell based ELISA [ 20 ]; (Fig. 7 ). Thus, PJ-S21 is an inhibitor of both X4 and R5 virus binding to the cellular receptor CD4 and coreceptors CXCR4/CCR5. PJ-S21 also inhibited gp120 binding to PBMCs as determined by flow cytometry (Fig. 8 ). To confirm that PJ-S21 functions as a virus entry inhibitor, the complex was added to cells at time intervals before and after infection of cells by HIV-1 IIIB and BaL, respectively. Results shown in Fig. 9 demonstrate that PJ-S21 interferes with early steps of the virus replicative cycle. To be considered as a topical microbicide, PJ-S21 must be formulated to withstand storage in a tropical environment. Accelerated thermal stability studies revealed that a water suspension of PJ-S21 maintained only 4, 11, and 33%, respectively, of its original activity (measured by inhibition of gp120-CD4 binding) when stored for 30 min at 60°C, and one week at 50°C or 40°C. On the other hand, a dried PJ-S21 powder remained fully active after storage at 50°C for 12 weeks (the longest time used in the evaluation). Consequently, anhydrous formulations should be preferred for further development. Three such formulations were prepared: two kinds of suppositories, melting at 37°C, and a tablet (for compositions see Methods section). The inhibitory activity of PJ-S21 was fully preserved after 12 weeks storage at 50°C within tablets, and at 30°C within the suppositories (the highest temperature considered to prevent melting). Data showing the inhibition of infection by HIV-1 IIIB and BaL respectively, by PJ-S21 and its formulations (except the tablets which also contain anti-HIV-1 inhibitors other than PJ-S21, i.e. Carbopol 974P [ 18 ]) are summarized in Fig. 10 . Their inhibitory activities against HIV-1 IIIB and BaL were similar, unlike the inhibitory activities of the original PJs (Fig. 1 ). These formulations were also virucidal, albeit at concentrations higher than those sufficient for inhibition of infection. These experiments also revealed that PJ-S21 was not cytotoxic under the experimental conditions used. The inhibitory/virucidal activities were maintained in the presence of seminal fluid (SF) at a 1:1 (w/w) ratio of SF to PJ-S21; (data not shown). A microbicide can be considered potentially successful, only if it has antiviral activity against primary virus isolates belonging to distinct virus clades and phenotypes. PJ-S21 meets this requirement since it inhibited infection by primary HIV-1 strains of all clades tested having R5 and X4R5 (= dual-tropic) phenotypes (Table 1 ). Discussion Pomegranates have been venerated for millennia for their medicinal properties and considered sacred by many of the world's major religions. In deference to pomegranates, the British Medical Association and several British Royal Colleges feature the pomegranate in their coat of arms. The Royal College of Physicians of London adopted the pomegranate in their coat of arms by the middle of the 16 th Century [ 40 ]. The best known literary reference to the contraceptive power of pomegranate seeds is classical Greek mythology. Persephone (IIερσεφονη) had eaten six pomegranate kernels (from which juice is derived) while in the Underworld and for that many months the land remained infertile during the Fall and Winter (Fig. 11 ). Ironically, this report shows that pomegranate juice contains HIV-1 entry inhibitors targeted to the virus envelope corresponding to a class of anti-retroviral drugs still scarce in development [ 41 ]. PJ contains several ingredients [ 42 , 43 ] which, isolated from natural products other than PJ, were reported to have anti-HIV activity, for example: caffeic acid [ 44 ], ursolic acid [ 45 ], catechin and quercetin [ 46 , 47 ]. However, these compounds, in purified form, obtained commercially, did not block (at 200 μg/ml) gp120-CD4 binding as measured by the ELISA described above and did not adsorb to corn starch, unlike the entry inhibitor(s) from PJ. In fact, the supernatant after treatment of PJ with starch, and removal of the entry inhibitors, retained anti-HIV-1 activity and also inhibited infection by herpes virus type 1, unlike the HIV-1 entry inhibitors which adsorbed onto starch. Thus, the antiviral activities in the supernatant appeared to be non-specific, and probably similar to those of extracts from pomegranate rind [ 48 , 49 ], and were not characterized further. Additional information [ 50 - 53 ] has revealed that the findings apply to crude extracts from pomegranate rind prepared at elevated temperatures under conditions which destroy the HIV-1 entry inhibitor described here. The inhibitor(s) interfering with gp120 binding to CD4 (Fig. 2 and 5 ) blocked additional sites on gp120 (Fig. 3 ) involved in interaction with the CXCR4/CCR5 coreceptors (Fig. 4 , 6 and 7 ). This was not completely expected and can be explained either by the presence of multiple inhibitors with distinct or overlapping specificities in PJ-S21 or by induction of gp120 conformational changes [ 54 ] resulting in blockade of both CD4 and CXCR4/CCR5 binding sites on gp120. Similar effects have been noticed for other small molecule inhibitors [ 55 ]. Simultaneous blocking of more than a single site on HIV-1 involved in virus entry is expected to increase the effectiveness of candidate microbicides [ 11 ]. The target sites for the inhibitor(s) are likely to be located within the protein moiety of gp120 since binding of labeled Galanthus nivalis lectin (specific for terminal mannose residues [ 56 ]; and other lectins to gp120 oligosaccharides was not diminished in the presence of PJ or PJ-S21 (data not shown). Blocking of CD4 binding sites on HIV-1 gp120 by monoclonal antibodies or a CD4-IgG2 recombinant protein has been shown to be sufficient to inhibit HIV-1 infection of human cervical tissue ex vivo [ 11 ] and in preventing virus transmission to macaque monkeys when applied vaginally [ 57 ]. Therefore, it seems likely that PJ-S21 will be similarly effective, an expectation which remains to be confirmed. The application of PJ-S21 as a topical anti-HIV-1 microbicide requires reasonable uniformity among batches produced at distinct times and locations. Similarities in gp120-CD4 binding inhibitory activity among distinct freshly prepared and commercial juices stored for unknown periods (Fig. 2 ) suggest that this should be feasible. Pasteurization of juice for 30 seconds at 85°C resulted in complete loss of inhibitory activity. A commercial PJ concentrate exposed to 61°C, and two other concentrates, presumably prepared by evaporation at elevated temperatures, had no or drastically diminished activity. The gp120-CD4 inhibitory activity from PJ3 (juice with fructose and citric acid added), failed to bind to starch. Separate experiments revealed that these compounds interfere with inhibitor binding to corn starch. Therefore, PJs intended for production of the PJ-S21 complex must be sterilized by filtration and be free of additives. Particular attention must be devoted to the selection of starch, a pharmaceutical excipient generally used in vaginal formulations [ 58 ], for effective binding of the virus entry inhibitors from PJ. Among a dozen starches tested, the best results have been obtained with S21. With other brands, the adsorption of the inhibitors was either incomplete or their binding did not result in a complex having activity in the ELISA measuring gp120-CD4 binding inhibition (ARGO ® corn starch), presumably, because of irreversible binding of the PJ inhibitors. Interestingly, there are only a few references available regarding the use of starch as an adsorbent for different compounds: flavors [ 59 , 60 ], dyes [ 61 - 63 ], low-molecular mass saccharides [ 64 ], lipids [ 65 , 66 ], proteins [ 67 ] and iodine [ 68 ]. The intended dose of PJ-S21 for vaginal application is 1.0 to 1.5 g, (= 3.17 – 4.76 mg solids from PJ adsorbed onto starch) i.e. ≥ 100-fold higher than the dose needed for blocking HIV-1 infection in vitro (Fig. 10 , Table 1 ), and thus expected to meet requirements for likely in vivo protection against vaginal challenge [ 69 ]. This quantity of PJ-S21 is produced from 5 to 7.5 ml of PJ, i.e. ≤ 5% of a single (150 ml) serving of juice, attesting to the safety, feasibility and economy of this proposed candidate topical microbicide. In an alternative approach to formulation development, PJ-S21 can be incorporated into a water dispersible film (similar to the widely available "breath control" strips) or into water dispersible sponges [ 70 ] which are converted into a gel following topical application [ 19 ]. Each of the above formulations would meet the following requirements: (1) minimization of waste disposal problems associated with the use of applicators needed for delivery of microbicidal gels/creams; (2) simplicity; (3) small packaging and discretion related to purchase, portability and storage; (4) low production costs; (5) amenability to industrial mass production at multiple sites globally and (6) potential application as rectal microbicides. Furthermore, it would remain possible to produce for local use PJ-S21 based gel formulations with a limited shelf life, avoiding the costs of producing dry PJ-S21 powders via appropriate low temperature drying processes. Whichever of these formulations is selected, adequate quality control will be needed to assure uniform anti-HIV-1 activity of the final product(s) and to establish reproducible conditions for manufacture. Conclusions PJ-S21 can be classified as an AAAA candidate microbicide: Acceptable; Accessible; Affordable; and Accelerative in transition from development to marketing. Thus, PJ-S21 would be expected to circumvent some problems associated with antiretroviral drugs and possibly some of the other candidate microbicides, i.e. uncertainty related to potential side effects, investment and time needed to establish inexpensive large scale production, and monopoly of supply. Abbreviations used AIDS, acquired immunodeficiency syndrome; BSA, bovine serum albumin; ED 50(90) , effective dose(s) for 50% (90%) inhibition of infection; ELISA, enzyme linked immunosorbent assays; FBS, fetal bovine serum; FLSC, a full length single chain protein consisting of BaL gp120 linked with the D1D2 domains of CD4 by a 20 amino acid linker; HIV-1, human immunodeficiency virus type 1; HRP, horseradish peroxidase; LTR, long terminal repeat; PBMCs, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PEG, polyethylene glycols; PJ, pomegranate juice; S21, PURITY ® 21 corn starch NF grade; SF, seminal fluid. Competing interests The authors declare that they have no competing interests. Authors' contributions ARN developed the concepts representing the basis of the manuscript and designed most experiments. NS contributed to the development of experimental techniques and carried out experiments other than infectivity assays. YYL did all the tissue culture work and viral infectivity assays. AKD did all the molecular modeling studies and contributed to the development of cell based enzyme linked immunosorbent assays. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC533885.xml
517940	The relationship between educational level and bone mineral density in postmenopausal women	Background This study describes the influence of educational level on bone mineral density (BMD) and investigating the relationship between educational level and bone mineral density in postmenopausal women. Methods A total of 569 postmenopausal women, from 45 to 86 years of age (mean age of 60.43 ± 7.19 years) were included in this study. A standardized interview was used at the follow-up visit to obtain information on demographic, life-style, reproductive and menstrual histories such as age at menarche, age at menopause, number of pregnancies, number of abortions, duration of menopause, duration of fertility, and duration of lactation. Patients were separated into four groups according to the level of education, namely no education (Group 1 with 209 patients), elementary (Group 2 with 222 patients), high school (Group 3 with 79 patients), and university (Group 4 with 59 patients). Results The mean ages of groups were 59.75 ± 7.29, 61.42 ± 7.50, 60.23 ± 7.49, and 58.72 ± 7.46, respectively. Spine BMD was significant lower in Group 1 than that of other groups (p < 0.05). Trochanter and ward's triangle BMD were the highest in Group 4 and there was a significant difference between Group 1 and 4 (p < 0.05). The prevalence of osteoporosis showed an inverse relationship with level of education, ranging from 18.6% for the most educated to 34.4% for the no educated women (p < 0.05). Additionally, there was a significant correlation between educational level and spine BMD (r = 0.20, p < 0.01), trochanter BMD (r = 0.13, p < 0.01), and ward's BMD (r = 0.14, p < 0.01). Conclusions The results of the study suggest that there is a significant correlation between educational level and BMD. Losses in BMD for women of lower educational level tend to be relatively high, and losses in spine and femur BMD showed a decrease with increasing educational level.	Background Osteoporosis has recently been recognized as a major public health problem by some governments and health care providers. In the European community, the number of men and women aged 65 years of older will increase steadily and the most dramatic changes will occur in the very elderly, in whom the incidence of osteoporotic fracture is greatest [ 1 ]. As the populations gets older, morbidity, mortality and financial costs attributed to osteoporosis are expected to rise. The economic costs related to osteoporotic fractures are substantial and will almost certainly increase further unless effective preventive interventions are widely implemented [ 2 ]. Peak bone mass is achieved soon after puberty, and bone is lost with various "insults", including ageing and postmenopausal changes. Factors influencing peak bone mass and loss range from nutrition, to lifestyle, to certain medical disorders. Educational level may also have an effect on bone mineral density since there is relationship between educational level and reproductive factors such as pregnancy and lactation and other lifestyle factors [ 3 - 7 ]. In developed countries a higher prevalence of most chronic diseases has been recognized among lower socio economic levels and in less educated subjects [ 8 - 14 ]; however, only a few and conflicting data are available for osteoporosis [ 15 - 18 ]. Since many risk factors for osteoporosis, such as diet, deficiency of trace minerals, reproductive factors, inactivity and tobacco use, are lifestyle variables related to social and cultural background [ 18 - 25 ], the influence of formal educational level on bone mineral density [BMD], together with establishment of a relationship between formal educational level and bone mineral density in postmenopausal women are the main concern of this study. Patients were compared according to years of formal education. We used formal education because it may be regarded as a composite or surrogate variable for overall socioeconomic status [ 10 ], and level of education [years of completed education) allows comparison between countries more readily than other socioeconomic indicators [ 13 ]. Purpose of this study was to evaluate the influence of formal education on BMD and investigating the relationship between educational level and bone mineral density in the postmenopausal women. Methods In Department of Physical Medicine and Rehabilitation, of the total 701 consecutive women screened, 132 were excluded. This study was undertaken in Dicle University, Diyarbakir, Turkey. The study protocol was reviewed and approved by the Dicle University Ethics Committee, and informed consent was obtained from all participants. A detailed history was taken from each woman including relevant life-style parameters and risk factors, and their weight and height measurements were recorded. The following exclusion criteria were applied for further analyses: (1) fractures after the age of 25; (2) menopause before the age of 40; (3) amenorrhoea greater than 6 months; (4) chronic conditions affecting bone density; (5) any use of corticosteroids. A total of 569 postmenopausal women, at 45 to 86 years of age (mean age of 60.43 ± 7.19 years) were considered. BMD of the spine and hip (neck, trochanter, and ward's triangle) were measured by dual-energy x-ray absorptiometry (NORLAND, 6938CE, New York, USA). According to the WHO [ 26 ] osteoporosis was defined as a lumbar BMD value more than 2.5 SD below the T-score, corresponding to 0.759 g/cm 2 [ 27 ]. The variation coefficient for consecutive determinations on spine and femur images in our laboratory was 1.9% at the lumbar spine and 1.6 % at the femur region. All spinal scans were reviewed for evidence of vertebrae with collapse or focal sclerosis by an experienced radiologist. In order to standardize the procedure, the patients all answered the same specially developed questionnaire supervised by the doctor (revised from the MEDOS Form) [ 28 ]. A standardized interview was used at the follow-up visit to obtain information on demographic, life-style, reproductive and menstrual histories such as age at menarche, age at menopause, number of pregnancies, number of abortions, duration of menopause, duration of fertility, and duration of lactation. The level of education is categorized in four groups according to the number of school years and the highest qualification received; no education (Group 1 = 209 patients), elementary (8 years or less, Group 2 = 222 patients), high school (9–11 years, Group 3 = 79 patients), and university (12 years or more, Group 4 = 59 patients). Body mass index (BMI; weight / height 2 ) was obtained through height and weight measurements by using a wall-mounted ruler and a digital scale. Recent dietary calcium intake (past 12 months) was assessed using standardized food models to estimate portion sizes [ 24 ]. Dietary calcium intakes were analyzed in two groups as inadequate (<500 mg/day) and adequate (500–1000 mg/day) [ 25 ]. The number of drinks consumed per week in the past 30 days, was used as the measure of current alcohol consumption (never use, very rare, frequently). Women who had smoked at least ten cigarettes per day during the five postmenopausal years were classified as smokers [ 25 ]. All patients classified, in terms of their reported current and life long smoking, into such group: 1) never use, 2) less than 1 packet, 3) 1–2 packet, and 3) more than 2 packets per day. They were also classified, in terms of their reported current and life-long caffeine use, into such groups: 1) never use, 2) 2 or below cup caffeinated coffee per day, 3) 3 or above cups caffeinated coffee per day. Physical activity is assessed by inquiring about the reported number of 20-min sessions of leisure-time physical activity per week and physically active behavior is defined as participation in more than two sessions per week; job-related physical exercise is not taken into account. Statistical analyses The statistical analyses were carried out with the SPSS/ PC-program. Differences in proportions for categorical variables were tested by chi-square test. The data are expressed as means ± SD. Statistical significance was tested using one way-ANOVA test and post-hok Bonferroni test for comparison of different groups. Pearson correlation test were computed to measure the association between the variables studied. The statistical significant set if the p-value was less than 0.05. Results Their reproductive and demographic characteristics are shown in Table 1 . When comparing the adequate calcium intake, the most educated women showed a statistically significant higher percentage than that of the other groups (p < 0.05). The mean ages of groups were 59.75 ± 7.29, 61.42 ± 7.50, 60.23 ± 749, and 58.72 ± 7.46, respectively. There was no significant difference among all groups with respect to age, BMI, age at menarche, age at menopause, and duration of menopause (p > 0.05) (Table 2 ). Table 1 Values for various reproductive and personal characteristics among 569 women screened for osteoporosis according to education level Variable No Education (Group 1 = 209) Elementary (Group 2 = 222) High school (Group 3 = 79) University (Group 4 = 59) Menstrual cycle pattern, (%) Regular cycles 159 (76) 151 (68) 66 (84) 43 (73) Irregular cycles 50 (24) 71 (22) 13 (16) 16 (27) Menopausal status, (%) Natural 171 (82) 189 (85) 72 (91) 40 (69) Iatrogenic 38 (18) 33 (15) 7 (9) 19 (31) Premenopausal HRT, (%) Never use 195 (93) 195 (88) 59 (75) 51 (86) Ever use 14 (7) 27 (12) 20 (25) 8 (14) Postmenopausal HRT, (%) Never use 201 (96) 203 (91) 63 (79) 52 (88) Ever use 8 (4) 19 (9) 16 (21) 7 (12) Physical daily activity, (%) Childhood Inactivity 6 (3) 33 (15) 14 (18) 8 (14) Mild activity 100 (48) 118 (53) 59 (75) 32 (54) Serious activity 103 (49) 71 (32) 6 (7) 19 (32) Adolescent Inactivity 8 (4) 35 (16) 15 (19) 9 (15) Mild activity 60 (29) 105 (47) 54 (68) 19 (32) Serious activity 141 (67) 82 (37) 10 (13) 31 (53) Adult Inactivity 56 (27) 83 (37) 38 (48) 22 (37) Mild activity 94 (45) 97 (44) 36 (46) 21 (36) Serious activity 59 (28) 42 (19) 5 (6) 16 (26) Calcium intake, (%) Adequate 102 (49) 107 (48) 36 (46) 45 (76)* Inadequate 107 (51) 115 (52) 43 (54) 14 (24) Smoking, (%) Never use 203 (97) 185 (83) 59 (75) 40 (69) < 1 packet 4 (2) 18 (8) 6 (7) 9 (15) 1–2 packet 2 (1) 9 (4) 10 (13) 1 (2) > 2 packet - 10 (5) 4 (5) 8 (14) Coffee, (%) Never use 117 (56) 102 (46) 43 (54) 20 (34) 2 or below cup 88 (42) 85 (38) 20 (25) 23 (39) 3 or above cup 4 (2) 35 (16) 16 (21) 16 (26) Alcohol, (%) Never use 203 (97) 204 (92) 64 (81) 40 (68) Very rare 2 (1) 12 (5) 10 (13) 14 (24) Frequently 4 (2) 6 (3) 5 (6) 5 (8) *Significant different from other groups (p < 0.05). Table 2 Comparison of reproductive characteristics of 569 women according to education level No Education Elementary High school University Age (years) 59.75 ± 7.29 61.42 ± 7.50 60.23 ± 7.49 58.72 ± 7.46 Body Mass Index 27.18 ± 5.14 28.23 ± 5.63 27.72 ± 6.14 28.45 ± 5.56 Age at menarche (years) 13.77 ± 1.36 13.75 ± 1.28 14.09 ± 0.83 13.22 ± 1.38 Age at menopause (years) 45.26 ± 5.57 45.22 ± 6.72 47.25 ± 4.69 45.38 ± 5.25 Duration of fertility (years) 31.48 ± 5.73 31.47 ± 6.79 33.25 ± 4.81 32.16 ± 5.61 Duration of menopause (years) 14.49 ± 7.8 13.20 ± 8.17 12.02 ± 8.02 12.41 ± 8.73 Number of abortions 1.31 ± 1.51 b, c 0.94 ± 1.64 0.51 ± 1.17 0.19 ± 0.54 Number of pregnancies 7.11 ± 3.38 a, b, c 4.93 ± 3.61 d, e 2.27 ± 1.91 2.29 ± 1.39 Duration of lactation (months) 133.2 ± 54.3 a, b, c 93.62 ± 50.66 d, e 45.76 ± 38.83 57.77 ± 35.36 Values are shown as mean ± standard deviation (SD) for all variables. a Significant different from elementary group, b Significant different from high school group, c Significant different from university group, d Significant different from high school group, and e Significant different from university group (p < 0.05). Number of abortions was higher in group 1 and 2 than those of group 3 and 4 (p < 0.05). There was no significant difference with respect to number of pregnancies and duration of lactation between group 3 and 4 while there was a significant difference among other groups (p < 0.05), and number of pregnancies and duration of lactation were found to be the highest in Group 1 and 2. Number of pregnancies and duration of lactation in Group 1 were 7.11 ± 3.38 and 133.23 ± 54.34 months and in Group 2 were 4.93 ± 3.61 and 93.62 ± 50.66 months (Table 2 ). Spine BMD was significant lower in Group 1 than that of other groups (p < 0.05). Trochanter and ward's BMD were the highest in Group 4 and there was a significant difference between Group 1 and 4 (p < 0.05). The prevalence of osteoporosis showed an inverse relationship with level of education, ranging from 18.6% for the most educated to 34.4% for the no educated women (p < 0.05) (Table 3 ). Table 3 Comparisons of bone mineral density of 569 women according to education level No Education Elementary High school University L2-4 BMD 0.62 ± 0.29 a, b, c 0.74 ± 0.19 0.77 ± 0.17 0.76 ± 0.33 Femoral Neck BMD 0.64 ± 0.19 0.61 ± 0.24 0.62 ± 0.24 0.64 ± 0.21 Trochanter BMD 0.49 ± 0.18 c 0.53 ± 0.19 0.56 ± 0.16 0.61 ± 0.12 Ward's triangle BMD 0.45 ± 0.17 c 0.49 ± 0.18 0.49 ± 0.20 0.58 ± 0.10 Osteoporosis (%) 72 (34.4) a, b, c 62 (27.9) d, e 17 (21.5) 11(18.6) Values are shown as mean ± standard deviation (SD) for all variables except percentage of osteoporosis. a Significant different from elementary group, b Significant different from high school group, c Significant different from university group (p < 0.05). Additionally, there was a significant correlation between educational level and spine BMD(r = 0.20, p < 0.01), trochanter BMD (r = 0.13, p < 0.01), and ward's BMD (r = 0.14, p < 0.01) but wasn't neck BMD (r = -0.02, p > 0.05). Discussion The health care costs, morbidity and mortality excess related to osteoporotic fractures are a major health problem in western countries [ 29 , 30 ]. In order to reduce these medical, social and economic burdens, which are expected to rise in forthcoming years, there is a need for preventive strategies based on health promotion campaigns [ 31 ]. To change health behavior related to modifiable risk factors for osteoporosis and to design targeted and more effective health messages [ 32 ], the programs have to take into account the socioeconomic and cultural background of the population strata in which the risk for osteoporosis is particularly prominent [ 15 ]. Although mechanisms of association between education and osteoporosis remain partly unexplained, most of the risk factors examined have shown distinct trends according to educational level. Although educational level may be an imperfect measure for socio economic status, many studies have clearly established that this marker acts as a good predictor not only for most chronic diseases [ 10 , 11 , 14 ] but also for many related risk factors [ 22 , 23 ]. In a study by La Vecchia et al., they found that education is a strong determinant of several chronic conditions, and the pattern of health care utilization also varied extensively according to education [ 11 ]. Varenna et al. evaluated 6160 postmenopausal women referred for their first densitometric evaluation and they found age at menarche, past exposure to oral contraceptives, prevalence of chronic diseases, physical activity, overweight and smoking showed significant trends according to years of education [ 15 ]. Also, as they had a cohort of postmenopausal women as the study group, they could show differences in the prevalence of osteoporosis among educational classes and the protective role played by increases in formal education. The present study showed that there was no significant difference among all groups with respect to age, BMI, age at menarche, age at menopause, and years since menopause. But there were statistically significant differences among groups in respect to number of pregnancies, duration of lactation, bone mineral density and percentage of osteoporosis. The comparison with studies performed in other countries can be misleading since eating habits are strongly influenced by ethnic and geographical backgrounds [ 7 ]. The meaning of the lower calcium intake observed in the least educated women could be referred to a real difference, taking into account the low sensitivity of the questionnaire used to assess calcium intake. During pregnancy and lactation the growing fetal and neonatal skeletons make major demands for calcium, respectively. There is good evidence now that during lactation a substantial part of this calcium demand is mobilized from the maternal skeleton even despite high dietary calcium. This effect could be especially important with multiple pregnancies and extended lactation. Magnus et al. undertook a random sample of 1514 Norwegian women and men to investigate knowledge of osteoporosis and attitudes towards methods for preventing this disease, and they concluded that in both men and women, increased knowledge of osteoporosis was correlated to a high level of education [ 33 ]. In several studies, authors have found that reproductive history has an inverse relation to bone density [ 3 - 6 , 34 - 41 ]. The bone density is adversely affected by both high rate of live birth and long period of breast feeding, common in the region where this study was carried out. The lower birth rate and short period of breast feeding found with the group having university or high school degree, may suggest that both birth rate and the breast feeding period may be associated with educational level. Furthermore, the calcium intake in the group with highest educational level was also found to be considerably higher than that of the other groups. The higher BMD values found with the group of highest educational level, may be attributed to the sufficient amount of calcium intake as was the case with this group Though the effect of formal educational level on bone mineral density has not yet been well established, the above findings may suggest some hypothetical comments. The findings of this study imply that osteoporosis which is related to bone mineral density, may be related to the educational level and the risks due to higher birth rate, excessive breast-feeding and insufficient calcium intake, and may be controlled through an improvement in educational level. Because of several limitations, caution must be exercised in interpreting the results of our study. Except for densitometric assessment, the results depend on self-reports. Even though self-report diagnoses have been shown to be valid [ 6 ], the level of formal education could bias the report about habits and health practices. Moreover, the sample was not randomly selected and it cannot be considered representative of postmenopausal women in Turkey. Similar studies are recommended to be carried out in different communities in an effort to confirm whether these findings can be generalized or yield a more complete insight into pathogenetic mechanisms. The knowledge of which population strata may be at greater risk of osteoporosis should be considered carefully for the purpose of health care planning and preventive strategies, making it possible to design tailored and culturally appropriate public health intervention programs. The protective role played by educational level, which increases with the years of formal education, could be due to other overall determinants can be indirectly inferred from our data, such as a better health status, a more positive attitude to taking drugs and a more efficient use of health care resources. All these determinants can be considered in the light of a greater concern by the women about their own health status, probably related to a different impact of health promotion messages. Conclusions In conclusion, the results of the study suggest that there is a significant correlation between educational level and BMD, and shows differences in the prevalence of osteoporosis among educational classes and the protective role played by increases in formal education. Losses in BMD for women of lower educational level tend to be relatively high, and losses in spine and femur BMD showed a decrease with increasing educational level. Although mechanisms of association between education and low bone mineral density remain partly unexplained, most of the risk factors examined have shown distinct trends according to educational level. Competing interests None declared. Financial competing interests None declared. Authors' contributions AG participated in the design of the study and performed the statistical analyses. AJS conceived of the study, and participated in its design and coordination. KN and RC participated in the sequence alignment and screened of subjects. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC517940.xml
538753	Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq	Background Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. Results The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases . Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25Δ crtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase . These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. Conclusion This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.	Background Forskolin, a labdane diterpene, is a major active compound isolated from tuberous roots of Coleus forskohlii Briq. (Lamiaceae) [ 1 ]. C. forskohlii has been used as an important folk medicine in India. Futher, forskolin has been found to be a potent activator of adenylate cyclase [ 2 ], leading to an increase in levels of c-AMP, which affects heart action, blood and intraocular pressure. Recently, forskolin has become commercially available as a drug for treating heart disease in Japan. Forskolin is not available by chemical synthesis due to its complicated structure. However, two groups have reported successful total synthesis of forskolin [ 3 , 4 ]. Isoprenoids are essential for the normal growth and development processes in all living organisms. Isopentenyl diphosphate (IPP; C 5 ) is a common metabolic precursor of all isoprenoids. Recently, several groups have demonstrated that two distinct pathways synthesise IPP in plants. The mevalonate (MVA) pathway occurs in the cytoplasm, and an alternative mevalonate-independent (2C-methyl-D-erythritol 4-phosphate; MEP) pathway occurs in plastids [ 5 - 7 ]. Geranylgeranyl diphosphate (GGPP) synthase catalyses the consecutive condensation of an allylic diphosphate with three molecules of IPP to produce GGPP, an essential linear precursor for biosynthesis of diterpenes, carotenoid, retinoids and side chain of chlorophyll [ 8 ]. GGPP synthase is an important branch point prenyltransferase enzyme in terpenoid biosynthesis. GGPP synthase genes have been cloned in a number of organisms including; Arabidopsis thaliana [ 9 , 10 ], Taxus canadensis [ 11 ], Helianthus annuus [ 12 ], Scoparia dulcis and Croton sublyratus [ 13 ], Sulfolobus acidocaldarius [ 14 ], Neurospora crassa [ 15 ], and mouse and human [ 16 ]. Amino acid sequence comparison has shown that GGPP synthases contain several domains of conserved amino acid residues including the first aspartate-rich motifs (FARM) and the second aspartate-rich motif (SARM) [ 17 ]. Futhermore, recent studies suggested that two amino acids at the four and five positions before FARM in the sequence, as well as an insertion in FARM of plant GGPP synthases play important roles in product length determination [ 13 , 18 ]. Carotenoids arise from the coupling of two molecules of GGPP. The carotenoid biosynthetic gene cluster ( crt genes) of Erwinia uredovora was elucidated [ 19 ], and is currently used to investigate the function of carotenoid related genes in a heterologous system. This crt gene cluster is composed of six genes; crtB (phytoene synthase), crtE (GGPP synthase), crtI (phytoene desaturase), crtX (zeaxanthin β-glucosidase), crtY (lycopene cyclase) and crtZ (β-carotene hydroxylase). Consequently, the production of carotenoids using E. coli harbouring the crt gene cluster can be used for the determination of GGPP synthase activity. GGPP synthase is suggested to be a key enzyme in the biosynthesis of forskolin. Herein, we report the cDNA encoding C. forskohlii GGPP synthase and its heterologous expression in E. coli . Results and discussion cDNA cloning and sequencing of C. forskohlii GGPP synthase gene The open reading frame (ORF) for full-length GGPP synthase gene encodes a protein of 359 amino acids, 1,077 nucleotides long, with a calculated molecular mass of 39.3 kDa. The amino acid sequence of C. forkohlii GGPP synthase revealed high homology throughout the entire coding region of Catharanthus roseus (75%), Arabidopsis thaliana (73%), Sinapis alba (72%), Croton sublyratus (69%), Scoparia dulcis (67%) and Mentha piperita (64%) (Fig. 1 ). However, comparison of the amino acid sequence with that of prokaryotic GGPP synthases showed a low level of homology (30–53%). Highly conserved residues were designated as domains I-VII. Two conserved aspartate-rich motifs, DDXX(X)D, were identifed. FARM and SARM have been shown to be important in substrate binding and catalysis [ 20 - 22 ]. Transient expression of putative localization signal of C. forskohlii GGPP synthase in tobacco cells Sequence alignment of plant GGPP synthases showed that the N-terminal region has a low level of homology. It is reasonable to assume that these GGPP synthases have localization signals in their N-terminal regions to target them into specific subcellular compartments. The N-terminal region of C. forskohlii GGPP synthase was predicted to be localized in chloroplasts by the ChloroP 1.1 Prediction Server. In an effort to determine the localization of C. forskohlii GGPP synthase, the sequence coding for the 80 amino acid sequence at the N-terminus of C. forskohlii GGPP synthase was fused to the N-terminus of the GFP reporter gene and transformed into BY-2 tobacco cells. The pattern of putative localization signal of C. forskohlii GGPP synthase was identical to the positive chloroplast targeting signal [35SΩ-pt-sGFP(S65T)] (Fig. 2 ). The N-terminal region of C. forskohlii GGPP synthase was determined to contain a chloroplast localization signal. Recently, plant GGPP synthases have been determined to be translocated into plastids, mitochondria and cytosol [ 9 , 23 ]. Heterologous expression and activity of C. forskohlii GGPP synthase In order to express C. forskohlii GGPP synthase , the gene was constructed and cloned into the plasmid pBluescript II KS - . The fusion protein of GGPP synthase with lacZ had a calculated molecular mass of 41.6 kDa, was observed in the soluble fraction of E. coli carrying pGGPPS after IPTG induction (Fig. 3 ). Functional activity of expressed GGPP synthase was investigated by genetic complementation with the carotenogenic crt gene cluster. Carotenoids are produced in E. coli harbouring a crt cluster gene from E. uredovora . Replacements of a crt gene with an unknown gene with the same activity, can be used to determine the function of the gene [ 15 ]. Herein, the C. forskohlii GGPP synthase gene was cloned into pBluescript II KS - vector (pGGPPS) in order to produce a lacZ fusion protein. pGGPPS was then transformed into E. coli DH10B carrying the plasmid pACCAR25Δ crtE in which the crtE encoding GGPP synthase had been deleted. The yellow color of carotenoid was observed in the transformant, indicating that pGGPPS carried the gene substituting the function of the crtE gene (Fig. 4 ). Carotenoid production of the transformants was compared with that of E. coli transformant carrying plasmid pACCAR25Δ crtE and pBAA encoding mouse GGPP synthase (positive control) [ 16 ], and with transformant carrying plasmid pACCAR25Δ crtE and a pBluescript II KS - (pBS) vector (negative control). This result suggested that the coding region of a cDNA of C. forskohlii GGPP synthase encodes a functional GGPP synthase. Expression of GGPP synthase gene in organs of C. forskohlii The expression of GGPP synthase gene was investigated by RT-PCR in different organs of C. forskohlii . Total RNA extracted from the roots, stems and leaves of an eight-month-old plant were analysed. The C. forskohlii GGPP synthase gene was strongly expressed in the leaves, whereas expression was decreased in stems and barely expressed in roots (Fig. 5 ). Therefore, the leaves are thought to be the primary location for forskolin synthesis. We previously reported the forskolin concentration in clonally propagated plant organs of C. forskohlii [ 24 ]. Tuberous roots and the stem base were determined to contain a higher concentration of forskolin than the organs. Moreover, the stem base, parts of the epidermis and cortex, the vascular bundle, and the pith were analysed separately. The highest concentration of forskolin was identified in the vascular bundle tissue. From these data, we proposed that GGPP synthase involved in biosynthesis of forskolin, is mainly synthesised in leaves, subsequently distributed to stems and finally accumulated in stem bases and roots. Forskolin production via non-mevalonate pathway In an effort to investigate the forskolin biosynthesis pathway by a non-mevalonate pathway, various concentrations of fosmidomycin, the specific inhibitor of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) enzyme in the non-mevalonate pathway were applied to the C. forskohlii culture and the forskolin content of roots was determined (Fig. 6 ). Treatment led to a decrease in forskolin, whereas 10 μM fosmidomycin had no effect on forskolin production. At higher concentrations a dose-dependent inhibitory effect was observed. At 1000 μM fosmidomycin, the forskolin content was decreased by up to fifty percent in comparison to the control tissue without inhibitor treatment. Thus, forskolin was thought to be synthesised via a non-mevalonate pathway. A recent 13 C-glucose feeding experiment using 13 C-NMR analytical methodology suggested the biosynthetic pathway of forskolin via a non-mevalonate pathway [ 25 ]. In addition, the DXR gene regarding the specific enzyme in the first step of the non-mevalonate pathway was cloned from C. forskohlii [ 26 ]. Conclusions C. forskohlii GGPP synthase was cloned and its subcellular localization was determined. The N-terminal region contained a signal which was localized in chloroplasts. Functional expression of GGPP synthase was investigated by genetic complementation with the carotenogenic crt gene cluster. Carotenoids were produced when the crtE gene was replaced with C. forskohlii GGPP synthase. GGPP synthase is thought to be involved in biosynthesis of forskolin, which is primary synthesised in the leaves, subsequently distributed to stems and finally accumulated in stem bases and roots. Methods Plant materials and reagents C. forskohlii plantlets were cultured in hormone-free MS (Murashige and Skoog) medium at 25°C under a 16 hours light cycle. The light intensity was 3000 lux and the relative humidity was 60%. Shoot cuttings (10 mm in length) propagated by shoot tip culture were successively cultivated in vermiculite. BY-2 tobacco single cell suspension [ 27 ] was cultured in liquid modified LS (Leinsmaier and Skoog) medium supplemented with 0.2 mg l -1 of 2,4-D (2,4-dichlorophonoxy acetic acid) under dark conditions at 25°C on an orbital incubator. Restriction enzymes, ligase, and PCR-polymerase were purchased from Takara Shuzo Co., Ltd. (Tokyo, Japan) and Toyobo Co., Ltd. (Tokyo, Japan). Fosmidomycin (FR-3154) was purchased from Molecular Probes (Oregon, USA). Chemical reagents were purchased from Sigma Chemical Company (St. Louis, USA) and Nacalai Tesque Inc. (Tokyo, Japan) Bacterial strains and plasmids E. coli TOP10F' and E. coli DH10B carrying the plasmid pACCAR25Δ crtE were used in the present investigation. The pUC119 vector was used for cDNA cloning and sequencing. The pBluescript II KS - vector was used as a GGPP synthase expression plasmid. The 35SΩ-sGFP(S65T) plasmid was used as a green fluorescent protein (GFP) reporter plasmid. The pBI121 plant vector and Agrobacterium tumefaciens LBA4404 were used for transformation of GFP and GFP-fusion genes to plant cells. cDNA cloning and sequencing of C. forskohlii GGPP synthase gene Total RNA was prepared from roots of the C. forskohlii culture using the acid guanidium-phenol-chloroform extraction procedure [ 28 ]. Single strand cDNA was synthesised using an oligo-dT adapter primer, M-MLV reverse transcriptase and total RNA as template. Degenerate primers were designed based on highly conserved amino acid sequences of previously cloned genes encoding plant GGPP synthases [ 13 ]. A 470 bp cDNA fragment was amplified using a nested PCR with Taq DNA polymerase and degenerate primers A, B, C and D (Table 1 ). The 3' end of cDNA was amplified using 3' rapid amplification of cDNA ends (RACE) with gene specific primers I and J, and adapter primer F. A 522 bp product was obtained by nested PCR. For 5' RACE, the first strand cDNA was polyadenylated at its 5' end by terminal deoxynucleotidyl transferase. The first and second PCR were performed with specific primers G and H and adapter primers E and F. A 285 bp product was obtained. The entire coding region of 1,077 bp was amplified by nested PCR using specific primers K, L, M and N designed from 5' and 3' RACE products. All amplified cDNA fragments were purified and digested with restriction enzymes at sites introduced via the PCR primers, and cloned into the vector pUC119. After transformation to E. coli TOP10F', clones harboring inserts were sequenced using a Model 310 Genetic Analyzer (PE Biosystems) using a BigDye Terminator Cycle Sequencing Kit. The amino acid sequence deduced from the nucleotide sequence was compared with sequence databases in the Genome Net WWW server using the FASTA program. Multiple amino acid sequence alignment was performed using the CLUSTALW Multiple Sequence Alignment in the GenomeNet CLUSTALW Server. Construction and expression of putative localization signal of C. forskohlii GGPP synthase A 240 bp fragment of the N-terminal region of C. forskohlii GGPP synthase was PCR-amplified using primers P and Q and the PCR product was digested and cloned into the Sal I- Nco I site of the 35SΩ-sGFP(S65T) plasmid. 35SΩ-pt-sGFP(S65T) was used as the positive control for chloroplast targeting [ 29 , 30 ]. GFP, GGPP synthase -GFP fusion and pt-GFP fusion with CaMV35SΩ promoter and NOS3' terminator [35SΩ-sGFP (S65T), 35SΩ- GGPP synthase -sGFP (S65T) and 35SΩ-pt-sGFP (S65T), respectively] were subcloned into the Hind III- EcoR I site of the pBI121 vector and then transformed into A. tumefaciens LBA4404. The transformants were cultured at 28°C for two days in YEB liquid medium containing 25 μg/ml of kanamycin and 25 μg/ml of rifampicin. The transformants were washed twice and re-suspended in YEB medium. Agrobacterium transformants (10 8 cells) were applied to four ml of five-day-old BY-2 suspension culture. The culture was incubated at 28°C for two days under dark conditions. GFP and GFP fusion protein were analysed by fluorescence microscopy using Nikon Eclipse TE2000-U model. Cells were observed at a 400 × magnification. Construction of plasmid for C. forskohlii GGPP synthase expression The coding region of a cDNA of C. forskohlii GGPP synthase was amplified by PCR using specific primers M and O. A PCR product was digested; purified and cloned into the Kpn I -Sal I site of pBluescript II KS - vector, namely pGGPPS. This plasmid was transformed into E. coli XL1-Blue MRF' for over-expression. The transformants were cultured in LB liquid medium containing 50 μg/ml of ampicillin and 25 μg/ml of chloramphenicol. The culture was induced with 1 mM isopropyl-1-thio-β-D-galactoside (IPTG) and incubated for six hours at 37°C. The cells were harvested and washed with 50 mM Tris-HCl pH 8.0 by centrifugation. The pellet was re-suspended, lysozyme was added and the mixture was incubated for 30 minutes. The mixture was then sonicated for four cycles of 15 seconds at one minute intervals. The soluble fraction was obtained after centrifugation at 10,000 × g for 10 minutes. SDS-PAGE was conducted in order to detect the proteins [ 31 ]. Genetic complementation expression The pACCAR25Δ crtE plasmid contains the gene cluster crtB , crtI , crtX , crtY and crtZ encoding carotenoid biosynthetic enzymes with the exception of crtE (encoding GGPP synthase). The plasmid pBAA containing mouse GGPP synthase (positive control plasmid) and E. coli DH10B carrying the plasmid pACCAR25Δ crtE was provided by Dr. M. Kawamukai, Shimane University, Japan [ 16 ]. pBluescript II KS - vector, pBS, was used as negative control. pGGPPS, pBAA and pBS were transformed into E. coli DH10B carrying the plasmid pACCAR25Δ crtE . All transformants were plated on LB agar medium containing 50 μg/ml of ampicillin and 25 μg/ml of chloramphenicol and then incubated for two to three days at 25°C. Reverse transcriptase-PCR (RT-PCR) An eight-month-old C. forskohlii was analysed in twelve separate parts; leaf (L1–L4), stem (S1–S5) and root (R1–R3). The numbering is based on the maturation of organs. Total RNA was extracted from each part of plant. One microgram of total RNA was used as the template for the synthesis of the first strand cDNA (using SuperScript First-Strand Synthesis System for RT-PCR, Invitrogen). Primers M and O, the first strand cDNA and KOD-polymerase were used for the amplification of C. forskohlii GGPP synthase with the condition of denaturation, 98°C, 15 seconds; annealing, 60°C, 2 seconds and extension, 74°C, 5 seconds. The 18S rRNA fragment used as an internal control was amplified using primers R and S under the same conditions of C. forskohlii GGPP synthase amplification. The amplified PCR products were analysed by 1.0% agarose gel electrophoresis. Analysis of forskolin production C. forskohlii plantlets were treated with various concentrations of fosmidomycin and then investigated for forskolin content using the HPLC method as previously described [ 26 ]. Forskolin was detected by comparison with the retention time of a forskolin standard (Sigma) detected by UV absorption at 202 nm. List of abbreviations crt , carotenogenic gene; FARM, first aspartate-rich motif; GFP, green fluorescent protein; GGPP, geranylgeranyl diphosphate; MEP, 2C-methyl-D-erythritol 4-phosphate; MVA, mevalonate; SARM, second aspatate-rich motif Authors' contributions SE carried out the molecular genetic studies, participated in the sequence alignment, forskolin analysis and drafted the manuscript. TF participated in the design of the study and coordination. MK participated in genetic complementation and coordination. YS conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC538753.xml
519004	DNA Barcoding: Promise and Pitfalls	null	In this issue of PLoS Biology, Hebert et al. (2004) have set out to test the resolution and performance of “DNA barcoding,” using a single mtDNA gene, cytochrome c oxidase I (COI), for a sample of North American birds. Before turning to details of this study, it is useful as context to consider the following questions: What is DNA barcoding, and what does it promise? What is new about it? Why is it controversial? What are the potential pitfalls? Put simply, the intent of DNA barcoding is to use large-scale screening of one or a few reference genes in order to (i) assign unknown individuals to species, and (ii) enhance discovery of new species ( Hebert et al. 2003 ; Stoeckle 2003 ). Proponents envisage development of a comprehensive database of sequences, preferably associated with voucher specimens representing described species, against which sequences from sampled individuals can be compared. Given the long history of use of molecular markers (e.g., allozymes, rDNA, and mtDNA) for these purposes ( Avise 2004 ), there is nothing fundamentally new in the DNA barcoding concept, except increased scale and proposed standardization. The former is inevitable. Standardization, i.e., the selection of one or more reference genes, is of proven value in the microbial community and in stimulating large-scale phylogenetic analyses, but whether “one gene fits all” is open to debate. Why, then, all the fuss? Initial reactions to the DNA barcoding concept have ranged from unbridled enthusiasm, especially from ecologists ( Janzen 2004 ), to outright condemnation, largely from taxonomists (e.g., see the February 2003 issue of Trends in Ecology and Evolution ). The former view reflects a real need to connect different life history stages and to increase the precision and efficiency of field studies involving diverse and difficult-to-identify taxa. The criticisms are mainly in response to the view that single-gene sequences should be the primary identifier for species (“DNA taxonomy”; Tautz et al. 2002 ; see also Blaxter 2004 ). At least for the macrobiota, the DNA barcoding community has moved away from this to emphasize the importance of embedding any large-scale sequence database within the existing framework and practice of systematics, including the importance of voucher specimens and of integrating molecular with morphological characters. Another point of contention—that DNA barcodes have limited phylogenetic resolution—arises from confusion about the scope of inference. At best, single-gene assays can hope to identify an individual to species or reveal inconsistencies between molecular variation and current perceptions of species boundaries. DNA barcoding should not be confused with efforts to resolve the “tree of life.” It should connect with and benefit from such projects, but resolving phylogeny at scales from species to major eukaryotic clades requires a very different strategy for selecting genes. Indeed, the very characteristic that makes the COI gene a candidate for high-throughput DNA barcoding—highly constrained amino acid sequence and thus broad applicability of primers ( Hebert et al. 2003 )—also limits its information content at deeper phylogenetic levels (e.g., Russo et al. 1996 ; Zardoya and Meyer 1996 ; Naylor and Brown 1997 ). Finally, while superficially appealing, the very term DNA barcoding is unfortunate, as it implies that each species has a fixed and invariant characteristic—like a barcode on a supermarket product. As evolutionary biologists, we should question this analogy. In evaluating the promise and pitfalls of DNA barcoding, we need to separate the two areas of application: molecular diagnostics of individuals relative to described taxa, and DNA-led discovery of new species. Both are inherently phylogenetic and rely on a solid taxonomic foundation, including adequate sampling of variation within species and inclusion of all previously described extant species within a given genus. Accurate diagnosis depends on low intraspecific variation compared with that between species, such that a short DNA sequence will allow precise allocation of an individual to a described taxon. The extensive literature on mtDNA phylogeography ( Avise 2000 ) indicates that this condition often holds, although there are exceptions. Furthermore, within many species there is sufficient structure that it will be possible to allocate an individual to a particular geographic population. Such identifications should be accompanied by a statement of confidence—e.g., node support in a phylogenetic analysis and caveats in relation to the breath of sampling in the reference database (e.g., whale forensics; Palumbi and Cipriano 1998 ). DNA-led species discovery is more contentious, but again is not new. In animals, inclusion of mtDNA evidence in biogeographic and systematic analyses often reveals unexpected diversity or discordance with morphology, which then prompts re-evaluation of morphological and ecological characteristics and, if warranted, taxonomic revision. But, despite recent proposals ( Wiens and Penkrot 2002 ; Hebert et al. 2004 ), it does not follow that mtDNA divergence should be a primary criterion for recognizing species boundaries (see also Sites and Marshall 2003 ). Potential limitations of using mtDNA to infer species boundaries include retention of ancestral polymorphism, male-biased gene flow, selection on any mtDNA nucleotide (as the whole genome is one linkage group), introgression following hybridization, and paralogy resulting from transfer of mtDNA gene copies to the nucleus. These are acknowledged by Hebert et al. (2004) and well documented in the literature ( Bensasson et al. 2001 ; Ballard and Whitlock 2004 ), including that on birds ( Degnan 1993 ; Quinn and White 1987 ; Lovette and Bermingham 2001 ; Weckstein et al. 2001 ). More specifically, using some level of mtDNA divergence as a yardstick for species boundaries ignores the low precision with which coalescence of mtDNA predicts phylogenetic divergence at nuclear genes ( Hudson and Turelli 2003 ). An additional problem with focusing on mtDNA (or any other molecular) divergence as a primary criterion for recognizing species is that it will lead us to overlook new or rapidly diverged species, such as might arise through divergent selection or polyploidy, and thus to conclude that speciation requires long-term isolation. For example, a recent mtDNA analysis of North American birds ( Johnson and Cicero 2004 ) showed that numerous avian species have low divergences and that speciation can occur relatively rapidly under certain circumstances. We contend, therefore, that whereas divergent or discordant mtDNA sequences might stimulate taxonomic reassessment based on nuclear genes as well as morphology, ecology, or behavior, mtDNA divergence is neither necessary nor sufficient as a criterion for delineating species. This view accords with existing practice: taxonomic splits in North American birds typically are based on multiple lines of biological evidence, e.g., morphological and vocal differences as well as genetic data ( American Ornithologists' Union 1998 ). We turn now to the core of Hebert et al.'s paper—COI sequencing of a substantial sample of North American birds (260 of 667 species) and its validity as a test of the barcoding concept. Their aim is to test “the correspondence between species boundaries signaled by COI barcodes and those established by prior taxonomic research.” North American birds are an interesting choice because their species-level taxonomy is relatively well resolved and there has been extensive previous analysis of levels of mtDNA sequence divergence within and among described species ( Klicka and Zink 1997 ; Avise and Walker 1998 ; Johnson and Cicero 2004 ). Herbert et al. (2004) found differences in COI sequences “between closely related species” that were 19–24 times greater in magnitude than the differences within species (7.05%–7.93% versus 0.27%–0.43%, respectively). From these data, they conclude that most North American bird species can be discriminated via molecular diagnosis of individuals and propose a “standard sequence threshold” of ten times the mean intraspecific variation (yielding a 2.7% threshold in birds) to flag genetically divergent taxa as “provisional species.” Thus, their analysis seeks to address both potential applications of DNA barcoding. Although Herbert et al. sampled a large number of species, a true test of the precision of mtDNA barcodes to assign individuals to species would include comparisons with sister species—the most closely related extant relatives. This would require that all members of a genus be examined, rather than a random sample of imprecisely defined close relatives, and that taxa be included from more than one geographic region. Johnson and Cicero (2004) showed the importance of comparing sister species when examining genetic divergence values in North American birds, with results that contrast strongly with those of Hebert et al. as well as previous studies (e.g., Klicka and Zink 1997 ). For 39 pairs of avian sister species, mtDNA sequence divergences ranged from 0.0% to 8.2%, with an average of 1.9% (cf. 7% to 8% among closely related species in Hebert et al.). Of these, 29 pairs (74%) are at or below the 2.7% threshold proposed by Herbert et al. and thus would not be recognized as species despite biological differences. Moreover, although only a few of these 39 pairs (see Table 1 in Johnson and Cicero [2004] ) had sufficient sampling to assess intraspecific variation in mtDNA sequences, these typically showed paraphyly in mtDNA haplotypes. Therefore, there are still too few cases with adequate sampling of intraspecific diversity for sister species pairs to know how common paraphyly is, although a recent meta-analysis found that 17% of bird species deviated from mtDNA monophyly ( Funk and Omland 2003 ). Collectively, these observations cast doubt on the precision of DNA barcoding for allocating individuals to previously described avian species. Empidonax flycatchers, which are renowned for their morphological similarity and could thereby benefit from DNA-based identification tools, provide an example of the importance of a more detailed analysis. A complete molecular phylogeny for this group ( Johnson and Cicero 2002 ) yielded distances between four pairs of sister species that ranged from 0.7% ( E. difficilis versus E. occidentalis ) to 4.6% ( E. traillii versus E. alnorum ); notably, the genetic distance between mainland and island populations of E. difficilis ( E. d. difficilis and E. d. insulicola , 0.9%) was greater than that between sister species ( Johnson and Cicero 2002 ). Herbert et al.'s analysis included only two species of Empidonax ( E. traillii and E. virescens ), which are not sisters but members of divergent clades. Because E. virescens is genetically distant from all other species of Empidonax (10.3% to 12.5% uncorrected distance; Johnson and Cicero 2002 ), its comparison with E. trailli therefore inflates estimates of interspecific distances within the genus. Another key point of Hebert et al.'s analysis was to estimate levels of intraspecific diversity. For 130 species of the 260 examined, more than two individuals were sequenced ( n = 2 to 12 individuals per species, mean = 2.4), and pooled pairwise genetic distances were found to be uncorrelated with geographic distances, leading Hebert et al. to conclude that “high levels of intraspecific divergence in COI in North American birds appear uncommon.” However, this makes the assumption that there is a common underlying pattern of phylogeographic structure, which is unlikely for North American birds ( Zink 1996 , Zink et al. 2001 ). If there is significant variation, assessment of intraspecific diversity can be based on a small sample of individuals only if individuals are sampled across existing population subdivisions for which geography and phenotypic variation are reasonable initial surrogates. The analyses presented by Hebert et al. will certainly stimulate further debate (a reply by Hebert et al. to the present letter is posted at http://www.barcodinglife.com ), but, for the reasons outlined here, they are not yet a definitive test of the utility of DNA barcoding for either diagnosis of individuals or discovery of species. We also question whether the results for North American birds can be extrapolated to the tropics, where DNA barcoding could have maximum value. In general, among-population sequence divergence increases with decreasing latitude, even excluding previously glaciated regions ( Martin and MacKay 2004 ), and studies of intraspecific genetic diversity in Neotropical birds have revealed a higher level of phylogeographic subdivision compared to temperate species ( Remsen 1997 , Lovette and Bermingham 2001 ). Thus, the general utility of mtDNA barcoding across different biogeographic regions—and between resident versus migratory taxa—requires further scrutiny. There is little doubt that large-scale and standardized sequencing, when integrated with existing taxonomic practice, can contribute significantly to the challenges of identifying individuals and increasing the rate of discovering biological diversity. But to determine when and where this approach is applicable, we now need to discover the boundary conditions. The real challenge lies with tropical taxa and those with limited dispersal and thus substantial phylogeographic structure. Such analyses need to be taxonomically broad and need to extend beyond the focal geographic region to ensure that potential sister taxa are evaluated and can be discriminated. There is also the need to examine groups with frequent (possibly cryptic) hybridization, recent radiations, and high rates of gene transfer from mtDNA to the nucleus. Only then will the skeptics be satisfied.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC519004.xml
479041	Cellular and Genetic Analysis of Wound Healing in Drosophila Larvae	To establish a genetic system to study postembryonic wound healing, we characterized epidermal wound healing in Drosophila larvae. Following puncture wounding, larvae begin to bleed but within an hour a plug forms in the wound gap. Over the next couple of hours the outer part of the plug melanizes to form a scab, and epidermal cells surrounding the plug orient toward it and then fuse to form a syncytium. Subsequently, more-peripheral cells orient toward and fuse with the central syncytium. During this time, the Jun N-terminal kinase (JNK) pathway is activated in a gradient emanating out from the wound, and the epidermal cells spread along or through the wound plug to reestablish a continuous epithelium and its basal lamina and apical cuticle lining. Inactivation of the JNK pathway inhibits epidermal spreading and reepithelialization but does not affect scab formation or other wound healing responses. Conversely, mutations that block scab formation, and a scabless wounding procedure, provide evidence that the scab stabilizes the wound site but is not required to initiate other wound responses. However, in the absence of a scab, the JNK pathway is hyperinduced, reepithelialization initiates but is not always completed, and a chronic wound ensues. The results demonstrate that the cellular responses of wound healing are under separate genetic control, and that the responses are coordinated by multiple signals emanating from the wound site, including a negative feedback signal between scab formation and the JNK pathway. Cell biological and molecular parallels to vertebrate wound healing lead us to speculate that wound healing is an ancient response that has diversified during evolution.	Introduction The capacity to heal wounds is essential for organisms to endure and thrive despite an occasionally hostile environment. Organisms throughout the animal kingdom can heal wounds, but mammalian wound healing has been studied most intensively because of its medical relevance. Wound healing must occur to restore health after trauma or surgery, or in conditions such as cancer or peptic ulcers in which internal processes cause tissue damage. Mammalian epithelial tissues display a characteristic set of responses to tissue damage, including the rapid formation of a blood clot at the site of injury, followed by spreading of the damaged epithelium across the wound gap to restore tissue integrity ( Martin 1997 ; Singer and Clark 1999 ). However, there are significant differences in the wound healing response depending on the specific tissue affected, its developmental stage, and the nature of the damage. For example, damaged fetal epidermis heals without leaving a scar ( Colwell et al. 2003 ), and a few adult tissues, including human liver, can regenerate large portions of the damaged tissue ( Diehl 2002 ). Some wounds, such as the common foot ulcers of diabetics, heal slowly or not at all ( Greenhalgh 2003 ), whereas others display an exaggerated response that results in disfiguring keloid scars ( Alster and Tanzi 2003 ). One important goal of wound healing research is to find ways to speed or alter the healing process. Another is to understand the fundamental cellular and molecular mechanisms by which cells sense tissue damage and signal to neighboring healthy cells to contain and repair it. Cellular studies of mammalian wound healing have shown that it is a complex process that takes weeks to complete and involves not just the damaged epithelial cells and their neighbors, but also fibroblasts and blood vessels in the underlying stroma, and inflammatory cells that are recruited to the wound site ( Martin 1997 ; Singer and Clark 1999 ). Only the first step in mammalian wound healing, the proteolytic cascade that culminates in fibrin deposition and clot formation, is well understood at the molecular level ( Furie and Furie 1992 ). As the clot forms, platelets bound to it and to the damaged tissue release additional procoagulant proteins as well as growth factors and chemokines that can attract neutrophils and monocytes that mediate an early inflammatory response. Keratinocytes at the wound margin become activated, break down their cell junctions, and assume a lamellipodial crawling morphology as they spread across the wound site to restore epithelial integrity ( Odland and Ross 1968 ; Clark et al. 1982 ). The early inflammatory cells release additional signals that can attract and activate fibroblasts, macrophages, and blood vessel endothelial cells. These cells infiltrate the wound site and form a specialized stroma called granulation tissue, which facilitates reepithelialization, helps contract the wound, and is later remodeled to form the scar. Although many different cell types are present at the wound site, and dozens of signaling molecules, receptors, matrix proteins, and proteases are known to be expressed during the healing process ( Martin 1997 ; Singer and Clark 1999 ), their roles in the process have been difficult to establish. This difficulty is due to the cellular and molecular complexity of wound healing and the challenges in manipulating wound gene expression and function in vivo. Hence, models of gene function in wound healing derive primarily from results of gene expression studies at wound sites, application of exogenous gene products to wounds, and studies in simple cell culture models such as keratinocyte monolayers. Analyses of wound healing defects in mouse knockouts of candidate genes have also begun to provide insight into the genes' roles in the process ( Werner et al. 1994 ; Romer et al. 1996 ). However, some of the genetic results challenge fundamental aspects of the prevailing models ( Ashcroft et al. 1999 ; Drew et al. 2001 ; Martin et al. 2003 ). The establishment of simpler, more tractable genetic systems to study wound healing could allow systematic genetic dissection of the process in vivo and complement studies in vertebrates and clinical settings. Over a half century ago, Wigglesworth demonstrated that the large hemipteran insect Rhodnius prolixus has a robust wound healing response ( Wigglesworth 1937 ). He characterized the response by light microscopy and described the proliferation and spreading of epidermal cells and the accumulation of blood cells (hemocytes) at the wound site. Since this pioneering work, only a few follow-up studies have appeared ( Lai-Fook 1966 , 1968 ). There has been little work on other insects aside from a number of studies of wound healing during imaginal disc and leg regeneration ( Reinhardt et al. 1977 ; Truby 1985 ; Bryant and Fraser 1988 ) and the recent discoveries that Drosophila embryos undergo a scarless wound healing process involving actin cable formation and filopodial extension ( Kiehart et al. 2000 ; Wood et al. 2002 ) and that wounded adult cells activate the Jun N-terminal kinase (JNK) signaling pathway ( Ramet et al. 2002 ; see below). Some attention has also focused on melanization, the formation of a heteropolymer of orthoquinones generated by phenoloxidase-catalyzed oxidation of mono- and diphenols ( Wright 1987 ) that accompanies certain infections, tumors, and wound healing ( De Gregorio et al. 2002 ; Ligoxygakis et al. 2002 ). We set out to investigate wound healing in Drosophila melanogaster because of the powerful genetic and genomic approaches available in this organism. These approaches have elucidated the molecular pathways that control many developmental and physiological processes. For example, genetic studies revealed a prominent role for a JNK signaling pathway in Drosophila dorsal closure, a developmentally programmed spreading of the embryonic epidermis ( Noselli and Agnes 1999 ). This process resembles epithelial spreading during vertebrate wound healing, and indeed this similarity and the expression patterns of JNK pathway transcription factors near wounds ( Verrier et al. 1986 ; Martin and Nobes 1992 ) prompted two recent genetic studies of JNK pathway activity in adult wound healing ( Ramet et al. 2002 ; Li et al. 2003 ). In this paper, we describe the cellular events and genetic requirements of epidermal wound healing in Drosophila larvae. A simple puncture wound assay was developed, and we use it to show that a plug rapidly forms at the wound site and subsequently melanizes to form a scab. We describe how epidermal cells surrounding the plug orient toward it and fuse to form a syncytium, and how the cells spread along and through the plug to reestablish epithelial continuity. We then use JNK pathway reporters and genetic analysis to demonstrate the induction and function of the JNK pathway in the process, and we use mutants that block scab formation, and a scabless wounding procedure, to elucidate the function of the scab. The results demonstrate that the cellular responses of wound healing are under separate genetic control, and that they are coordinated by multiple signals emanating from the wound site, including a negative feedback signal between scab formation and the JNK pathway. This establishes a tractable genetic system to study postembryonic wound healing, and the cellular and molecular parallels with vertebrate wound healing suggest that some of the fundamental steps in the process are evolutionarily conserved. Results A Larval Epidermal Wound Healing Assay A puncture wounding procedure was developed in which early third instar (L3) Drosophila larvae were lightly anesthetized and then stabbed with a 0.1-mm–diameter steel needle, about the size of six epidermal cells ( Figure 1 ; see also Figure 3 A). To ensure reproducibility, larvae were always stabbed at the dorsal midline halfway between the hair stripes of abdominal segments A3 or A4. Wounding did not cause a developmental arrest, because the wounded larvae continued to grow and pupariated 48 h after wounding, similar to mock-wounded controls ( Figure 1 B– 1 G), and 90% or more of the wounded larvae survived the procedure (see below). We then analyzed the major morphological, cellular, and molecular events of healing ( Figure 1 N) by visualizing wounds at different stages of healing in live and heat-killed whole-mount larvae, in histochemically or immunostained larval fillets, and in sections through wounds that we examined by transmission electron microscopy (TEM) (schematized in Figure 1 A). Figure 1 Scab Formation and Resolution during Puncture Wound Healing (A) Puncture wound assay. L3 larvae are punctured at the dorsal midline with a 100-μm diameter pin; they are then cultured and the healing wounds analyzed as shown. (B–E) Photomicrographs of heat-killed L3 larvae before wounding (B) and at the indicated times after wounding (C–E). Note larval growth during wound healing. Anterior is up. (F) L2 larva wounded as above and analyzed in L3, 60 h after wounding. Wounding in L2 allows visualization of late stages of wound healing without the complication of pupariation, which begins about 48 h after wounding in the standard L3 assay. (G) A mock-wounded L2 larva visualized 60 h after wounding. Note that it and the wounded larva (F) grew to a similar extent. (H–M) Close-up images of (B–G) showing unwounded cuticle (H and M) or wound sites (I–L, boxed regions in C–F) to show detail of scab. Micrographs are of living larvae taken shortly before the corresponding images of the whole heat-killed larvae above. Bar, 500 μm (for B–G), 50 μm (for H–M). (N) Timing of wound responses. Solid lines, time response was most often observed; dashed extensions to left, time response was occasionally observed; dashed extensions to right, time response was diminishing; BL, basal lamina. Figure 3 Epidermal Cell Orientation and Fusion around Puncture Wounds (A–E) w; UAS-GFP.nls/+; A58-Gal4/+ larvae that express GFP (green) in epidermal cell nuclei were mock-wounded (A) or puncture wounded (B–E), cultured for the indicated time, filleted open, fixed, and immunostained for Fasciclin III (red) to label the basolateral surface of the cells. (A) Pre-wounding. Dashed circle, size of the 100-μm pin used for wounding. (B) 2 h postwounding. Some cells at the wound margin have elongated and oriented toward the wound (arrowheads). (C) 8 h postwounding. Cells at the wound margin have begun fusing to form a syncytium. Note the syncytium with four nuclei that contains a partially degraded, radially-oriented membrane domain (arrow) and scattered puncta of Fasciclin III staining in the cytoplasm (arrowhead) that may be membrane breakdown intermediates. (D) 48 h postwounding. The central syncytium contains ten or more nuclei, some of which are located in extensions (arrowheads) that may represent recent fusions of peripheral cells with the central syncytium. Other peripheral cells have oriented toward the syncytium but not fused with it. (E) 60 h postwounding. A large syncytium with more than 30 nuclei. (F) 8 h postwounding. Larva was treated as above but immunostained for Coracle (red), a septate junction component. The central syncytium contains nine nuclei. Bar, 50 μm. Bleeding and Scab Formation at the Wound Site Unwounded larvae have a semitransparent white cuticular surface with rare or no blemishes (see Figure 1 B, 1 G, 1 H, and 1 M). Beneath the cuticle is the epidermis ( Figure 2 ), an epithelial monolayer that secretes the cuticle at its apical (external) surface and is lined by a basal lamina along its basal surface ( Figure 2 A, 2 C– 2 F). Immediately after puncture wounding, a variable amount of blood (hemolymph) escapes from the wound site (data not shown). Within 10–15 min, the wound site begins to darken (see Figure 1 C and 1 I) and a plug forms in the gap ( Figure 2 B and 2 G). The plug is composed of debris, presumably the remnants of necrotic cells damaged by wounding that are disorganized and highly vesiculated and not bound by a cell membrane or basal lamina ( Figure 2 H and 2 K). The plug may also contain blood coagulation products (see Discussion). Figure 2 Ultrastructural Analysis of Puncture Wound Healing (A) Schematic of unwounded epidermis showing the cell monolayer, its apical cuticle lining, and basal lamina. White ovals indicate nuclei. (B) Schematic of recently wounded epidermis showing a plug of cell debris in the wound gap. Cells and ruptured cuticle at the wound margin are shown. (C–S) TEM sections of unwounded (C–F) and wounded (G–S) larvae at the times indicated after wounding. Transverse sections through each wound site are shown (C, G, J, N, and Q) along with close-ups of the boxed regions at right. c, cuticle; d, debris; e, epidermis; ec, epicuticle; m, muscle; n, epidermal nucleus; p, plug; pc, procuticle; s, scab; t, trachea (C) Pre-wounding. The epidermis and cuticle are intact. (D) Apical surface of epidermal cell showing villi (arrowhead) that secrete cuticle. (E) Basal surface. Arrowhead, basal lamina. (F) Epidermal cuticle. The epicuticle (top three layers) overlies the striated procuticle layer. (G) 1 h postwounding. The epidermis and cuticle are discontinuous but the gap is filled with a plug (outlined by dashed line) of cell debris. The epidermis has partially separated from the cuticle beyond the wound margin (asterisks). (H) The plug contains highly vesiculated cell debris. (I) The epidermis separating (asterisk) from overlying cuticle appears vesiculated and is presumably necrotic. (J) 2 h postwounding. The outer portion of the plug has melanized to form an electron-dense scab (outlined by white dashed line). The epidermis and cuticle are still discontinuous. (K) Debris, including a necrotic trachea, in the plug. The plug is not bounded by a membrane or basal lamina. (L) Portion of scab showing melanized debris and trachea. (M) Close-up of a lamellipodium (bracket) extending into a plug at the outer edge of another 2-h wound. Note basal lamina (arrowhead) along the lamellipodium. (N) 8 h postwounding. The epidermis has migrated across the gap to reestablish continuity, and has secreted new cuticle beneath the scab. (O) A region of epidermal cell cytoplasm near wound plug debris contains vesiculated material (outlined by dashed line) that is probably phagocytosed debris. (P) The newly established epidermis under the wound has a continuous basal lamina (arrowhead) and apical villi (arrow) secreting cuticle. (Q) 24 h postwounding. The new cuticle underlying the scab is thicker and the scab is more electron dense. Four nuclei in close apposition are in a syncytium because there are no membranes separating them. (R) Portion of scab and old cuticle. Note that cuticle in contact with the scab is melanized. (S) Cytoplasmic extension (arrowhead) engulfing debris at the basal surface of the epidermis of another 24-h wound. Bar, 10 μm (C, G, J, N, and Q), 0.33 μm (D and E), 0.83 μm (F), 1 μm (H, M, and P), 2 μm (I and O), 1.67 μm (K and L), 4 μm (R), 1.25 μm (S). Over the next 24 h, the outer part of the plug is converted into a scab. This part of the plug becomes electron dense ( Figure 2 J and 2 L) as the scab enlarges and darkens (see Figure 1 D, 1 E, 1 J, and 1 K), presumably due to a melanization reaction. Melanization affects all of the external structures at the wound site including the debris, the edges of the damaged cuticle ( Figure 2 N, 2 Q, and 2 R), and even entrapped tissues such as tracheae ( Figure 2 L). By 2 or 3 d after wounding, debris is cleared, the scab resolves, and the exterior of the animal resumes a nearly normal appearance (see Figure 1 F and 1 L). Epidermal cells that grow back across the wound gap (see below) appear to participate in debris clearance, because they extend processes that engulf the debris ( Figure 2 S) and contain within their cytoplasm vesiculated material resembling debris ( Figure 2 O). Other components of the plug and scab may be degraded extracellularly or passively shed from the wound site. Epidermal Cells Orient toward the Wound and Fuse to Form a Syncytium The response of epidermal cells to wounding was examined in transgenic larvae in which epidermal cell nuclei were labeled with green fluorescent protein (GFP) and cell membranes were immunostained for the basolateral membrane marker Fasciclin III or the septate junction protein Coracle ( Figure 3 ). Epidermal cells at the wound site underwent two dramatic morphological changes in the several hours following wounding. First, beginning about a half hour after wounding, cells at the wound margin began to elongate and orient toward the wound, often tapering toward the wound site ( Figure 3 B). Second, these cells fused with each other to form a syncytium. Normally, epidermal cells are mononuclear ( Figure 3 A). However, as early as 1 h after wounding, the radially oriented plasma membrane domains (parallel to the long cell axis) began to break down as the circumferential domains joined, creating multinucleate cells around the wound. This can be seen in the Fasciclin III and Coracle stains, which showed incomplete ( Figure 3 C and 3 ) or absent ( Figure 3 C– 3 E) radial domains of plasma membrane staining; the loss of these membrane domains was sometimes accompanied by scattered puncta of staining in the cytoplasm, which may be membrane breakdown intermediates (see Figure 3 C). TEM analysis confirmed the absence of plasma membrane between epidermal nuclei beneath and adjacent to the wound site (see Figure 2 Q). Syncytia were nearly always present by 4 h after wounding. As healing progressed, the polarization and fusion of epidermal cells spread outward from the wound. As cells bordering the wound fused, the more-peripheral cells just beyond the syncytium began to polarize toward the wound (see Figure 3 D). Some of these cells apparently proceed to fuse with the central syncytium, because the average number of nuclei per syncytium increased over the 2 d following wounding, creating a large syncytium with as many as 30 nuclei surrounding the wound (see Figure 3 E). Epidermal cell or nuclear division do not contribute to growth of the syncytium, because neither was detected around the wound site or elsewhere in the epidermis by immunostaining for phosphorylated histone H3, a marker of condensed mitotic chromosomes, 4–24 h after wounding (data not shown). Highly asymmetrical syncytia like the one shown in Figure 3 D probably represent cases in which a subset of polarized peripheral cells had fused with the central syncytium. Peripheral cells may fuse to each other before fusing to the central syncytium, because satellite syncytia separate from the central syncytium were occasionally observed. Epidermal Cells Spread along and through the Plug to Reestablish Epithelial Integrity A key step in wound healing is the closure of the epidermal gap and reestablishment of epithelial integrity. By 2 h after wounding, the epidermis was still discontinuous but the breach was filled by the plug and developing scab (see Figure 2 J). Ultrastructural studies showed that during the next 6 h, as the epidermal cells oriented toward the puncture site and fused to form a syncytium, they also spread along and through the plug, led by lamellipodial extensions (see Figure 2 M), until epithelial continuity was reestablished (see Figure 2 N). No multicellular actin cable indicative of the “purse string” closure mechanism of embryonic wound healing ( Wood et al. 2002 ) was observed in the spreading cells (see Materials and Methods ). A thin basal lamina was present along the length of the lamellipodia (see Figure 2 M), suggesting that basal lamina is synthesized by the cells before or during their migration. Following reepithelialization, new cuticle was secreted (see Figure 2 N– 2 P). By 24 h after wounding, a thick new cuticle layer was present that was continuous with the old cuticle at the wound margin (see Figure 2 Q). Most of the wound plug debris ended up outside the new cuticle layer and eventually melanized to form scab (see Figure 2 Q and 2 R), although occasionally some was left beneath the epidermis and was later phagocytosed or degraded (see Figure 2 S). The JNK Pathway Is Activated in a Gradient and Promotes Reepithelialization To elucidate the genetic control and interdependence of the cellular events of wound healing, we investigated the activity and function of the JNK signaling pathway in the process ( Figure 4 ). The epidermal spreading in some ways resembles the epidermal spreading of dorsal closure, which depends on the JNK pathway. During dorsal closure, the mitogen-activated protein kinase kinase kinase kinase Misshapen ( Su et al. 1998 ) is activated, triggering a phosphorylation cascade that ultimately activates the JNK Basket ( Riesgo-Escovar et al. 1996 ; Sluss et al. 1996 ) . Basket phosphorylates the Drosophila Jun and Drosophila Fos transcription factors ( Riesgo-Escovar and Hafen 1997 ), thus inducing expression of puckered (puc), which encodes a phosphatase that negatively regulates Basket, and other targets ( Martin-Blanco et al. 1998 ). To test for JNK pathway activation in the larval puncture wound assay, we assayed expression of lacZ transcriptional reporters of puc and misshapen (msn), two genes induced by JNK pathway activation in other contexts ( Martin-Blanco et al. 1998 ; Ramet et al. 2002 ). Figure 4 Induction and Function of the JNK Pathway around Puncture Wounds (A–C) Larvae carrying the JNK pathway reporter puc-lacZ, which expresses a nuclear β-galactosidase, were mock-wounded (A) or puncture wounded (B and C), and then cultured for the indicated times before staining with X-gal to visualize reporter activity (blue). There is little reporter activity in unwounded epidermis (A), but 4 h after wounding the reporter is expressed in a gradient emanating from the wound, with highest expression in the row of epidermal nuclei at the wound margin and decreasing levels in surrounding nuclei out to five cell diameters away. At 24 h (C), reporter expression has declined. (D–F) Larvae carrying the JNK pathway reporter msn-lacZ, treated as above. Wounding-induced reporter expression is seen out to seven cell diameters. (G–I) Larvae carrying msn-lacZ and A58-Gal4 and UAS-bsk DN transgenes (to inactivate the JNK pathway in larval epidermis), treated as above. Reporter induction is inhibited, but the scab forms normally. (J and K) Larvae carrying msn-lacZ and either UAS-bsk DN alone as control (J) or A58-Gal4 and UAS-bsk DN transgenes (K), wounded as above and analyzed 24 h later by immunostaining for Fasciclin III and β-galactosidase. Reporter induction is inhibited in (K), but epidermal cells have oriented toward the wound, and although nuclear β-galactosidase staining is faint, careful inspection shows that the cells closest to the wound have fused to form a syncytium. Syncytium formation was confirmed using the A58-Gal4>UAS-GFP.nls marker. (L and M) Larvae carrying msn-lacZ and either UAS-bsk DN alone as control (L) or A58-Gal4 and UAS-bsk DN transgenes (M), wounded and analyzed 24 h later by TEM. Note that the epidermis in M has failed to spread across the wound gap and is still discontinuous (asterisks). No cuticle has been synthesized in the wound gap, but the cuticle flanking the wound appears thickened. Bar in (I), 100 μm (for [A–I]). Bar in (K), 50 μm (for [J and K]). Bar in (M), 5 μm (for [L and M]). In unwounded larval epidermis, there was little or no detectable expression of either the msn or the puc reporter ( Figure 4 A and 4 D). However, within 1 h after wounding, expression of both reporters was readily detected in epidermal cells surrounding the wound, and by 4 h both exhibited robust expression ( Figure 4 B and 4 E; unpublished data). The msn and puc reporters were induced in large, roughly symmetrical zones extending three to seven cell diameters out from the puncture site. Within each zone, the reporters were expressed in a gradient, with cells closest to the puncture site exhibiting the highest level of expression, suggesting that the reporters are induced by a signal emanating from the wound site. The zone of expression of the msn reporter was typically broader than that of puc, perhaps because it is more sensitive to the inducing signal. Expression of both reporters peaked between 4 and 8 h after wounding and declined thereafter, with expression restricting to cells closest to the wound ( Figure 4 C and 4 F). To determine the function of JNK pathway induction, we analyzed wound healing in larvae in which the JNK pathway was inactivated. Because null mutations in JNK pathway genes block dorsal closure and are embryonic lethal, we selectively inhibited the pathway in larval epidermis by expressing a dominant-negative form of Basket JNK (upstream activation sequence-basket dominant negative [UAS-bsk DN ]) under the control of the A58-Gal4 driver, an epidermal-specific driver that turns on early in larval development. UAS-bsk DN was used because it is the most potent JNK pathway inhibitor available (see Materials and Methods ): it gave a severe dorsal closure phenotype and lethality when expressed in the embryonic epidermis using e22c-Gal4 or 69B-Gal4 drivers. By contrast, larvae expressing UAS-bsk DN under control of the A58-Gal4 driver were viable and active and did not display any morphological abnormalities, suggesting that the JNK pathway does not play a critical role in the larval epidermis under normal environmental conditions. However, following wounding, induction of the msn reporter was almost completely abolished ( Figure 4 G– 4 I), and the wound healing process was dramatically affected. We analyzed the effect of JNK pathway inhibition on wound healing using the assays used for wild-type larvae. There were no detectable defects in the early steps in wound healing, including scab formation, epidermal cell orientation toward the wound, and epidermal cell fusion to form a syncytium (see Figure 4 G– 4 K). However, ultrastructural analysis showed that reepithelialization was blocked or defective, with no cytoplasmic processes or only extremely fine or distorted processes and no new cuticle synthesis beneath the scab 16 h after wounding ( Figure 4 L and 4 M; data not shown). To further test the requirement of the JNK pathway in reepithelialization, we analyzed larvae in which a portion of the epidermis was abraded by a nonpenetrating pinch wounding procedure (described further below) that leaves a much larger gap in the epidermis than does a fine puncture wound and hence provides a more rigorous test of wound reepithelialization ( Figure 5 ). In control larvae in which the JNK pathway was not inhibited, the epidermis spread to close the gap, and full reepithelialization was evident within 24 h after wounding ( Figure 5 A and 5 B). By contrast, in larvae in which the JNK pathway was inhibited, the epidermis did not spread, and a large gap remained ( Figure 5 C). We conclude that induction of the JNK pathway promotes spreading and reepithelialization of the larval epidermis but appears to be dispensable for other steps in wound healing, including scab formation, cell orientation, and cell fusion. Figure 5 Cellular Responses and Genetic Requirements of Pinch Wound Healing (A–D) Larvae carrying the msn-lacZ reporter and the indicated transgenes or mutations were pinched with a forceps to abrade a region of dorsal epidermis but leave the overlying cuticle intact. Wounded larvae were cultured for the indicated times and immunostained for Fasciclin III (red) and β-galactosidase (green). (A) 6 h after pinch wounding. Note the large epidermal gap (asterisk) at the wound site. Some cells at the wound margin have elongated and oriented toward the wound (arrowheads). Others have fused to form syncytia (arrow). (B) 24 h after pinch wounding. The epidermis has spread to close the gap. Note disorganization of epidermis and syncytia (arrows) at site of healed wound. (C) An A58-Gal4 and UAS-bsk DN larva 24 h after pinch wounding. Epidermal spreading is inhibited and a large wound gap remains (asterisk). However, cells at the wound margin still orient toward the wound (arrowheads) and fuse to form syncytia (arrows). (D) A hemizygous lz r15 mutant larva 24 h after pinch wounding. lz r15 blocks crystal cell development and scab formation at puncture wounds ( Figure 6 ), but no defects are observed in pinch wound healing. (E and F) Larvae carrying msn-lacZ reporter were mock-wounded (E) or pinch wounded (F), cultured for 4 h, and stained with X-gal (blue). Wounding induces reporter expression in a gradient extending out four cell diameters. The gap (asterisk) lacks a scab. Bar, 100 μM. Crystal Cells Promote Scab Formation To investigate the role of the scab in puncture wound healing, we sought ways to block scab formation genetically ( Figure 6 ). Crystal cells are a special type of blood cell that contain distinctive, crystal-like intracellular inclusions and have long been hypothesized to play a role in melanization responses such as those in scab formation ( Rizki and Rizki 1959 , 1984 ). The gene lozenge (lz) encodes a transcription factor required for development of the crystal cell lineage ( Lebestky et al. 2000 ), and crystal cells are severely reduced or absent in lz r15 homozygous or hemizygous larvae ( Figure 6 A and 6 B). The lz r15 mutant larvae failed to form a scab detectable by light microscopy ( Figure 6 C and 6 D), and TEM analysis showed a diffuse plug at the wound site instead of the consolidated, electron-dense plug and scab that are normally present 24 h after wounding ( Figure 6 E and 6 F). This defect in scab formation is likely due to the effect of lz r15 on crystal cells, and not some other effect of the mutation, because scab formation was also inhibited in larvae homozygous for Black cells (Bc) (data not shown), a mutation that alters crystal cell morphology and eliminates serum phenoloxidase activity ( Rizki et al. 1980 ). We conclude that crystal cells are required to consolidate and melanize the plug to form a scab during wound healing. Figure 6 Effect of lz on Scab Formation and the Other Events of Puncture Wound Healing (A and B) Posterior of lz + ( w 1118 ) (A) and lz r15 mutant (B) L3 larvae. Larvae were heated so crystal cells appear as tiny black dots. No crystal cells are apparent in the lz r15 mutant. Bar, 200 μm. (C and D) Micrographs of control lz + ( w 1118 ) (C) and lz r15 mutant (D) L3 larvae 4 h after puncture wounding. No scab is seen at the lz r15 wound site (encircled). Bar, 50 μm. (E and F) TEM sections through 24-h–old puncture wounds of a control lz r15 /+ heterozygote (E) and a hemizygous lz r15 mutant larva (F), both carrying the msn-lacZ transgene. A consolidated, electron-dense scab has formed in the control larva (E), but only a diffuse plug with peripheral electron density is present at the lz r15 hemizygous wound (F). The electron density of the lz r15 plug might be due to residual melanization activity in the lz r15 mutant. Although reepithelialization is complete in the lz r15 mutant wound, the epidermis contains large vacuoles and abundant apical processes, and it is separated by a gap (asterisks) from the old cuticle and has not secreted new cuticle. Other 24-h lz r15 mutant wounds analyzed had necrotic or discontinuous epidermis at the wound site (not shown). Bar, 10 μm. (G and H) Fluorescence micrographs of 20-h puncture wounds in control (G) and lz r15 mutant (H) larvae carrying the msn-lacZ reporter that were treated as above and immunostained for Fasciclin III (red) and β-galactosidase (green). Epidermal cells at both control and lz r15 mutant wounds have fused to form syncytia (arrows), and cells in the control are oriented toward the wound site (arrowheads). The orientation response of epidermal cells in the lz r15 mutant is difficult to assess because cell borders out to six cell diameters away from the wound appear slack and wavy. Bar, 50 μm. (I–L) X-gal stains of 6-h–old puncture wounds of control lz + (I and K) or lz r15 hemizygous mutant larvae (J and L) carrying either msn-lacZ (I and J) or puc-lacZ (K and L). Note the absence of scabs and the increase in reporter activity (blue) in lz r15 . The basal level of reporter expression in unwounded epidermis was not increased in lz r15 (not shown). Bar, 50 μm. Untreated lz r15 larvae were viable and active, but few survived the normal puncture wound procedure ( Figure 7 ). By 4 h after wounding, only 55% of lz r15 larvae were alive, and by 24 h only 15% survived, most of which were sluggish and flaccid. By contrast, 85% or more of the lz + control larvae survived the wounding procedure. Thus, scab formation is critical for healing puncture wounds. Figure 7 Effect of lz on Survival Following Puncture Wounding Control lz + ( w 1118 ) and lz r15 mutant larvae were puncture wounded or mock-wounded and cultured for 4 or 24 h. The percentage of treated larvae alive and motile at each time is shown. Values are the average (± standard error of the mean) of three to six independent experiments with ten or more treated larvae per time point. The Scab Stabilizes the Wound Site and Prevents Superinduction of the JNK Pathway We next investigated the cellular events of wound healing in lz r15 larvae, using the methods described above for wild-type and JNK pathway mutants, except that sharper pins were used for wounding to increase survival and allow analysis of the later stages of wound healing. Most of the cellular responses to wounding appeared to initiate in lz r15 mutants, although they did not progress normally. Epidermal cell fusion occurred, but the syncytium often occupied a greater area than in control larvae (see Figure 6 G and 6 H). The surrounding epidermal cells also appeared to organize around the wound, but their cell borders were slack and wavy, even several cell diameters out from the wound, making it difficult to assess whether they had oriented toward the wound (see Figure 6 H). A similar though less severe “wavy border” phenotype was observed in Bc mutant larvae. TEM analysis revealed that the epidermal cells up to 200 μm or more beyond the wound margin separated from the overlying cuticle around the wound (see Figure 6 F). However, the detached cells extended numerous fine cellular processes in an apparent attempt to close the wound. Sometimes the edges of the punctured epidermis met to restore epithelial integrity, but in most cases they did not (see Figure 6 F; data not shown). The lz r15 mutation also caused superinduction of the JNK pathway reporters. Although the basal expression level of the msn and puc reporters in unwounded epidermis was unchanged, both were expressed at higher levels and in an expanded zone around the wound site at 3, 6, and 24 h after wounding (see Figure 6 I– 6 L; data not shown). A similar effect was observed in Bc mutants. Thus, scab formation limits induction of the JNK pathway around puncture wounds. To further investigate the role of the scab in wound healing, a scabless wound healing procedure was developed. The larval cuticle was gently pinched with dissecting forceps, leaving the cuticle intact but abrading a patch of epidermal cells from its inner surface (see Figure 5 A). Although these pinch wounds did not bleed or form scabs, the epidermal cells at the wound site underwent many of the same responses seen at puncture wounds. Many cells at the wound margin oriented toward the wound, and some fused with neighboring cells to form syncytia (see Figure 5 A and 5 B). Also, the msn reporter was induced in a gradient in the cells surrounding the wound (see Figure 5 A and 5 F), and the cells spread to close the wound gap within 24 h (see Figure 5 B). Thus, each of the major epidermal cell responses to wounding can occur normally in the absence of a scab, provided the cuticle remains intact. Indeed, the primary function of the scab may be to restore integrity to the cuticle and wound site, because lz r15 mutant larvae did not display any defects in the healing of pinch wounds: epidermal cells around the wound polarized and fused like in lz + controls, the JNK pathway reporters were induced at their normal levels and in their normal domain around the wound site, and the epidermal cells spread across the wound and healed with normal kinetics (see Figure 5 D). Thus, the critical function of the scab appears to be to provide stability to the damaged cuticle and wound site, and the defects observed in the epidermal cell responses following puncture wounding of lz r15 mutants most likely arise secondarily to the persistent instability of the wound site. Discussion We established an epidermal wound healing assay in Drosophila larvae and elucidated the cellular events and genetic requirements of the healing process. Following puncture wounding, the damaged epidermal cells and their neighbors execute a series of responses that limit blood loss and restore integrity to the epidermis and overlying cuticle (see Figure 1 N). Shortly after wounding, a plug forms in the wound gap. Over the next several hours, the outer portion of the plug melanizes to form a scab, and epidermal cells at the wound margin begin to elongate and orient toward the wound. They then fuse with each other to form a syncytium surrounding the wound. Subsequently, more-peripheral cells orient toward and fuse with the central syncytium. No proliferation of epidermal cells or actin cable formation was detected at the wound site. Instead, the epidermal cells surrounding the wound migrate along or through the plug to restore continuity of the epithelium and its basal lamina and cuticle lining. Each of these responses—scab formation, epidermal cell orientation and fusion, and epidermal spreading and reepithelialization—occurs at characteristic times and positions during wound healing. However, our results suggest that these responses are under separate genetic control and are not contingently coupled ( Figure 8 ). Scab formation is dependent on crystal cells and is inhibited by the lz r15 and Bc mutations. Epidermal spreading and reepithelialization require bsk and JNK pathway activity, which is rapidly induced in epidermal cells surrounding the wound site. Epidermal cell orientation and fusion can proceed even in the absence of scab formation or JNK pathway activity. Although the different responses have distinct genetic requirements and can initiate independently of each other, we identified one important interaction between them. In lz r15 and Bc mutants, reepithelialization initiated but was not always completed, and the JNK pathway was hyperinduced, implying that the scab normally facilitates reepithelialization and restrains JNK activation. Figure 8 Model of the Cellular Events and Genetic Requirements of Larval Wound Healing Puncture wounding disrupts the epidermis and overlying cuticle and triggers the three parallel series of events shown, each with distinct genetic requirements. Plug and scab formation stabilize the wound site, which promotes epidermal cell spreading and suppresses JNK activation, perhaps by a negative feedback mechanism (dashed line). The lz and Bc genes promote scab formation, presumably by promoting crystal cell development and the production and secretion of serum melanization factors by these cells. The spreading epidermal cells synthesize cuticle and basal lamina, and they clear wound site debris by phagocytosis. Pinch wounding disrupts the epidermis but not the overlying cuticle and triggers only the events shown in black. However, cuticle and basal lamina synthesis and phagocytosis have not been examined in pinch wounds and are only inferred to occur from the puncture wound studies. Wounding may induce additional signals (not indicated) that attract blood cells (plasmatocytes) and tracheal branches. Below, we discuss the mechanisms and functions of each of these wound healing responses and the signals that trigger them, and suggest a mechanistic basis for the observed interaction between scab formation, reepithelialization, and JNK activation. We also compare wound healing in Drosophila with the related processes in mammals and speculate on their evolutionary relationship. Formation and Function of the Scab The wound plug that forms shortly after puncture wounding contains cell debris, and it may also contain blood coagulation products like those identified in other arthropods ( Nakamura et al. 1976 ; Barwig 1985 ; Geng and Dunn 1988 ) and recently in Drosophila ( Scherfer et al. 2004 ). Over the next few hours the plug rapidly darkens and becomes electron dense, presumably the result of a melanization reaction. Although the nature and extent of melanin cross-linking to tissues has not been studied, it seems likely that the polymer links to wound plug components and cuticle to strengthen and stabilize the wound site. Our results identify two important requirements for maturation of the plug and scab formation. One is crystal cells. The mutations lz r15 and Bc, which block crystal cell development or function, inhibited scab formation at puncture wound sites. The effect was particularly striking in lz r15 mutants: no scab was detected by light microscopy, and ultrastructural studies revealed only disorganized, amorphous debris where the scab normally forms. Because crystal cells are not commonly found at puncture wound sites (G. Fish, M. J. Galko, and M. A. Krasnow, unpublished data), these results support a model in which crystal cells promote scab formation by supplying serum factors such as prophenoloxidase that are necessary to form or consolidate the scab. The other critical requirement for scab formation is a breach spanning both the epidermis and cuticle. In both puncture and pinch wounds, the epidermal layer is disrupted, but only puncture wounds formed scabs. The most obvious difference between the two types of wounds is that the cuticle layer remains intact after pinch wounding. This leads us to propose that scab formation is initiated by a signal generated or liberated by cuticle rupture, or by contact between serum and ruptured cuticle or air. One consequence of this would be local activation of prophenoloxidase by serine proteases that are present as inactive zymogens in insect cuticle ( Ashida and Brey 1995 ; Jiang et al. 1998 ). The scab appears to serve at least three functions in wound healing. One is to prevent exsanguination. Drosophila has an open circulatory system, so any rupture of both epidermis and cuticle will lead to blood loss. lz r15 mutants did not form scabs and survived poorly after puncture wounding; the few surviving larvae appeared flaccid, suggesting continued blood loss from the wound. Although the wound plug likely provides a temporary stop to bleeding, scab formation appears necessary to form a stable hemostatic barrier. Second, the scab likely serves an immune function, which may also enhance survival upon puncture wounding. The orthoquinone precursors of melanin are cytotoxic to microorganisms ( Nappi and Ottaviani 2000 ) and may guard against infection even before the scab matures to form a physical barrier to microbe entry. The third function of the scab is to provide structural stability to the wound, which is critical for the next phase of wound healing, reepithelialization. This is inferred from the failure of reepithelialization following puncture wounding of lz mutants that are unable to form a normal scab. lz loss of function does not cause any intrinsic defect in reepithelialization, because reepithelialization of pinch wounds proceeded normally in the mutant. Also, the JNK pathway was activated in the wounded epidermis of lz mutant puncture wounds, and the cells at the wound margin appeared to initiate reepithelialization by extending processes into the wound gap. However, the epidermis did not always complete closure and seal the gap. These results suggest that when both epidermis and cuticle are disrupted, the scab is necessary to stabilize the wound gap to allow the epidermis to spread across and close it. In the absence of a scab, the JNK pathway is hyperinduced, epidermal cells at the wound margins separate from the overlying cuticle and extend abundant cytoplasmic processes, and a chronic wound ensues. Epidermal Cell Orientation and Fusion Two intriguing cellular responses during wound healing are the orientation of epidermal cells toward the wound site and their subsequent fusion to form a syncytium. During orientation, epidermal cells at the wound margin lengthen along the axis radial to the puncture site and contract along the axis circumferential to it, with the part of the cell closest to the wound contracting most, imparting a characteristic taper. These cells then fuse by joining their circumferentially-oriented plasma membrane domains and eliminating their radially-oriented membrane domains that contact neighboring cells. This implies that epidermal cells are able to sense their location with respect to the wound and organize their cytoskeleton and plasma membrane domains with respect to it. As wound healing proceeds, cell orientation and fusion typically spread to include more-peripheral cells, resulting in large syncytia with up to 30 nuclei at puncture wounds and smaller, scattered syncytia at pinch wounds. The occurrence of these responses in cells beyond the wound margin suggests that they are not a direct result of damage but rather are induced and oriented by a signal produced by wounding that can spread several cell diameters away from the wound. The function of epidermal cell orientation and fusion may be to fit more cells around the wound and help seal off the wound site by eliminating intercellular spaces. This may be similar to the fusion of mammalian macrophages into multinucleate giant cells as they surround and engulf large foreign bodies ( Chambers 1977 ). Indeed, like macrophages, the fusing epidermal cells appear to be phagocytically active, engulfing debris at the wound site. Although the close temporal and spatial relationship between epidermal cell orientation and fusion suggests that these responses are likely to be coupled, mutants that specifically block each process will be required to determine if they are contingently coupled or just coordinated by a common upstream signal. Epidermal Spreading and Reepithelialization The most important cellular response for the long-term health of the animal is the restoration of epithelial integrity. However, spreading of the epithelium does not usually manifest until several hours after wounding. This allows time to induce the JNK pathway and activate the cell migration machinery in the epidermal cells, and to assemble a mature wound plug through or along which the cells move. Spreading appears to be an active process of epidermal cell migration, as no evidence of a purse-string closure mechanism or cell division was detected during spreading; instead, the earliest morphological manifestation of spreading was lamellipodial extensions, a hallmark of active cell migration, that course along and through the wound plug. Spreading likely requires a shift in the adhesion properties of epidermal cells from their normal tight association with the overlying cuticle to an affinity for the plug, and an ability to burrow through the plug. Spreading also requires a signal at the wound site that induces the JNK pathway in surrounding cells and activates the cell migration machinery. This must be a local signal emanating from the wound site that can influence cells up to seven cell diameters away. The activating signal might guide the migrations across the wound gap, or the cells might spread randomly along the matrix until their movement is arrested by contact inhibition. The main function of reepithelialization is to restore the normal barrier function of the epidermis. Indeed, the spreading epidermal cells ultimately secrete a thick layer of cuticle at their apical surface that displaces the scab, and they also supply the new basal lamina. The spreading cells also appear to play an important role in clearing wound site debris, as they were occasionally seen engulfing debris and often contained material resembling debris in phagosomes. Epidermal cells may share this scavenging role with plasmatocytes, circulating phagocytes recruited to wound sites after wounding (G. Fish, M. J. Galko, and M. A. Krasnow, unpublished data). Once reepithelialization is completed, debris is cleared, and the scab is sloughed or degraded, it is difficult to discern the old wound site by light microscopy. However, healing is not scar-free; the syncytium formed during healing persists and marks the wound site at least until metamorphosis begins. Occasionally, such syncytia are also seen in untreated larvae; these may be scars of naturally occurring wounds suffered earlier in larval life. The Wound as a Signaling Center The results suggest that there are multiple signals induced by wounding that control and coordinate the different events of larval wound healing: a signal that initiates formation of the wound plug and scab, one that orients surrounding epidermal cells and induces them to fuse, one that activates the JNK pathway and epidermal cell migration, and one dependent on scab formation that suppresses the JNK pathway. There may also be signals that attract plasmatocytes to combat infection and tracheal branches to increase wound oxygenation (M. J. Galko, unpublished data). These signals have distinct properties. One obvious difference is their range of activation around the wound. The signal that triggers plug and scab formation does so only at the epidermal and cuticular breach, whereas the JNK pathway activator influences cells up to seven cell diameters away. Some signals influence only the damaged cells and their neighbors, whereas others like the putative plasmatocyte and tracheal attractants must reach circulating cells and other tissues. Some of the signals are likely to be diffusible molecules released by damaged cells. These could be intracellular components such as uric acid, histones, or heat shock proteins, all of which have been shown to be released by necrotic mammalian cells and are implicated as intercellular signals ( Ohashi et al. 2000 ; Li et al. 2001 ; Scaffidi et al. 2002 ; Shi et al. 2003 ). They could also be more conventional signaling molecules like the fibroblast growth factors secreted upon vertebrate wounding ( Werner et al. 1992 ). Not all signals need be freely diffusible. Surface-bound signals could be sequentially propagated from one cell to the next, and some signals might be mechanical rather than chemical. Wounding appears to alter the tensile properties of the epidermis around the wound site (see Figure 6 H), which could serve as a mechanical stimulus for some responses. This is an attractive idea for the control of JNK pathway activation, because changes in mechanical stress have been shown to activate JNK signaling in other cell types ( Ingram et al. 2000 ; Kippenberger et al. 2000 ; Martineau and Gardiner 2001 ). Once reepithelialization is complete, tension could be restored, and signaling would diminish. Indeed, such a feedback circuit provides a plausible mechanistic basis for the inhibitory effect of scab formation on the JNK pathway (see Figure 8 ). In the absence of a scab, reepithelialization fails and tension is not restored, leaving the JNK pathway unconstrained. It is not obvious how many distinct signals are generated by wounding, because individual signals might regulate multiple responses. A high priority now is to molecularly identify the signals and the mechanisms by which they control and coordinate the wound healing responses. Comparison with Other Wound Healing Processes The healing of larval puncture wounds bears little resemblance to wound healing in the developing embryo, which occurs rapidly via actin cable assembly and filopodial extension by cells at the wound margin, and proceeds without scab formation ( Kiehart et al. 2000 ; Wood et al. 2002 ). Despite the substantial structural differences between Drosophila and mammalian epidermis, embryonic wound healing appears similar to that in mammalian embryos, where it is also a rapid process involving actin cable formation but no apparent hemostatic or inflammatory response ( Martin and Lewis 1992 ). Likewise, larval wound healing displays many similarities to postembryonic wound healing in mammals. Both processes commence with formation of a plug or clot that fills the wound gap. Both use the plug as a provisional substratum through which surrounding epidermal cells migrate. In both processes, the surrounding epidermal cells orient toward the wound site, become activated for migration, and spread through the plug in a similar manner—by extending lamellipodia and then their cell bodies into the plug until epidermal continuity is reestablished. The cells then redifferentiate to restore epidermal morphology. In addition, inflammatory cells are recruited to the wound in both processes, and the plug is remodeled to form a scab that is degraded or sloughed when repair and redifferentiation are complete. Despite these general similarities, there are many specific differences between each parallel step in Drosophila and mammals. For example, the composition of the Drosophila plug and the mammalian clot probably differ, because clotting mechanisms in arthropods involve proteolytic cascades similar to those in mammals but different coagulogens ( Nakamura et al. 1976 ; Barwig 1985 ; Geng and Dunn 1988 ; Scherfer et al. 2004 ). Also, Drosophila epidermal cells near the wound do not proliferate during reepithelialization as do their mammalian counterparts ( Martin 1997 ). The cells surrounding a Drosophila wound fuse to form a syncytium, whereas mammalian epidermal cells remain distinct but dynamically rearrange their junctions with neighboring cells as they spread. Spreading Drosophila cells carry a basal lamina with them, whereas migrating mammalian epidermal cells detach from the basal lamina ( Odland and Ross 1968 ; Clark et al. 1982 ). The most important difference may be the extent of cell recruitment to the wound site and subsequent remodeling of the plug, which are substantial in mammals but limited in Drosophila . Evolution of the Wound Healing Response The similarities between Drosophila and mammalian wound healing responses prompt the question of whether these are homologous processes or the result of convergent evolution. Because there would likely have been strong selective pressure early in evolution for a wound healing response, we favor the idea that wound healing is an ancient process that evolved before the divergence of flies and mammals and subsequently diversified. Indeed, the parallels in the embryonic and postembryonic processes suggest that distinct embryonic and postembryonic wound healing mechanisms were already in place at the time of divergence. If this evolutionary hypothesis is correct, then there should still be common molecular manifestations in Drosophila and mammals of the ancestral processes. Actin cable formation in embryonic wound healing may be one such manifestation, and the induction of JNK signaling pathways and their involvement in reepithelialization of postembryonic wounds may be another ( Ramet et al. 2002 ; Li et al. 2003 ). Others may become apparent once the wound healing processes have been genetically dissected. The wound healing process described here, with its simple tissue architecture, streamlined response, and accessible genetics, provides a tractable system for identifying additional genes and fundamental mechanisms of wound healing. Materials and Methods Fly strains and genetics The mutant lz r15 is a molecular null allele ( Daga et al. 1996 ). Bc 1 is a dominant mutation that was used in the homozygous condition ( Rizki et al. 1980 ). The msn-lacZ allele was l(3)06946 ( Spradling et al. 1999 ) and the puc-lacZ allele was l(3)A251.1 ( Martin-Blanco et al. 1998 ); both are P[ lacZ, rosy + ] enhancer trap insertions in the respective loci that express a nuclear β-galactosidase; heterozygotes were used to monitor reporter activity. For analysis of msn-lacZ reporter activity in the lz r15 mutant background, lz r15 , FRT18E/Y; msn-lacZ/+ hemizygous male larvae were compared to lz r15 , FRT18E/white [w] 1118 ; msn-lacZ/+ heterozygous female siblings; similar comparisons were made for the puc-lacZ reporter. w 1118 was used as a control strain because most of the other strains employed carried a background w – mutation. The Gal4/UAS system ( Brand and Perrimon 1993 ) was used for protein misexpression. The A58-Gal4 driver expresses the yeast Gal4 transcription factor throughout the larval epidermis beginning in L1 (A. Ghabrial, M. J. Galko, and M. A. Krasnow, unpublished data); e22c-Gal4 ( Lawrence et al. 1995 ) and 69B-Gal4 ( Brand and Perrimon 1993 ) express Gal4 throughout the embryonic epidermis. UAS-GFP-actin ( Verkhusha et al. 1999 ) was driven by A58-Gal4 to visualize actin dynamics within the larval epidermis . UAS-GFP.nls ( Shiga et al. 1996 ) expresses a nuclear-localized GFP. UAS-bsk DN ( Adachi-Yamada et al. 1999 ), UAS-puc ( Martin-Blanco 1998 ), UAS-Jra.bZip ( Kockel et al. 1997 ), and UAS-kayak.bZip ( Zeitlinger et al. 1997 ) express different JNK pathway inhibitors. When crossed to the e22c-Gal4 or 69B-Gal4 drivers, only UAS-bsk DN and UAS-puc gave a strong dorsal closure defect like JNK pathway mutants. To express Basket DN in larval epidermis, female w 1118 , UAS-bsk DN , UAS-bsk DN /w 1118 ; msn-lacZ, A58-Gal4 /+ larvae (and sibling males lacking the w 1118 chromosome) were used. To express Puckered, w 1118 ; UAS-puc/ msn-lacZ, A58-Gal4 larvae were used. Larvae of the same genotypes but lacking A58-Gal4 served as controls. Wounding assays Animals were reared on standard cornmeal-dextrose fly media at 25 °C. L3 larvae were rinsed with water, lightly anesthetized with ether, and then visualized under a stereomicroscope and impaled with a 0.1-mm steel needle (Fine Science Tools, Foster City, California, United States) at the dorsal midline between the hair stripes of abdominal segment A3 or A4. Typically, the needle pierced through the larva but only the entry wound was analyzed. After wounding, larvae were rinsed and returned to fly media in 1-dram vials and cultured at 25 °C. For experiments depicted in Figure 7 , care was taken to select both larvae and wounding pins of uniform size because larval survival following wounding is significantly influenced by these variables. For pinch wounds, L3 larvae prepared as above were pinched with #5 dissecting forceps (Fine Science Tools) at midbody on the dorsal side for approximately 10 s and then cultured as above. Mock-wounded control larvae were prepared and cultured as above, except that needle impalement and pinching were omitted. Incisional wounds were not analyzed because incision caused early L3 larvae to burst and die. TEM Larvae were dissected at 4 °C in EM fixative (3% glutaraldehyde, 2% paraformaldehyde, and 2.5% dimethylsulfoxide in 0.2 M sodium phosphate buffer [pH 7.2]) and pinned ventral side up on a Sylgard (Dow Corning, Midland, Michigan, United States) surface. A ventral incision along the length of the animal was made with dissecting scissors, and the four corners of the epidermis were stretched with forceps and pinned to the surface. Internal tissues were removed, and the epidermis was fixed an additional 15 min at room temperature and then trimmed to a flat piece of epidermis surrounding the wound. Tissue samples were incubated for 1 h at 4 °C in 1% osmium tetroxide, stained overnight at 4 °C in 0.5% uranyl acetate, dehydrated through a graded series of ethanol concentrations and propylene oxide, and embedded in EMbed 812 (Electron Microscope Sciences, Hatfield, Pennsylvania, United States) with N, N-dimethylbenzylamine, which was polymerized overnight at 55 °C. Transverse sections (75–90 nm) were cut through the wound site with a Leica Ultracut ultramicrotome (Leica, Wetzlar, Germany) and collected on formvar/carbon-coated 75 mesh copper grids and stained for 20 s in supersaturated uranyl acetate:acetone (1:1) followed by 0.2% lead citrate for 3–4 min. Specimens were observed with an 80-kV beam on a JEOL TEM-1230 microscope (JEOL, Peabody, Massachusetts, United States), and images were captured on a Gatan Multiscan 791 digital camera (Gatan, Pleasanton, California, United States). For TEM analysis of lz r15 mutants, there was no scab to mark the wound site, so lz r15 , FRT18E/Y; msn-lacZ/+ larvae were used and stained with X-gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) (see below) to locate the wound. Wounded larvae were dissected in phosphate-buffered saline (PBS), fixed in 2% glutaraldehyde for 15 min at room temperature, and stained with X-gal as described below, except the staining solution lacked Triton X-100 and contained 20 mM K 4 [FeII(CN) 6 ], 20 mM K 3 [FeIII(CN) 6 ], 2 mM MgCl 2 , and 0.2% X-gal in PBS. The propylene oxide dehydration steps during TEM sample preparation were omitted to preserve the X-gal reaction product. Histochemistry and immunohistochemistry For β-galactosidase histochemistry, larvae carrying lacZ transgenes were dissected open in PBS, fixed for 15 min at room temperature with cold 2% glutaraldehyde, rinsed with PBS, and then stained at room temperature for 6 h (puc-lacZ) or 2 h (msn-lacZ) in 150 mM NaCl, 10 mM Na 2 HPO 4 , 3 mM K 4 [FeII(CN) 6 ], 3 mM K 3 [FeIII(CN) 6 ], 1 mM MgCl 2 , 0.1% Triton X-100, and 0.2% X-gal. For immunostaining, primary antibodies were anti-Coracle monoclonal antibodies 9C and C61516B ( Fehon et al. 1994 ) (1:500 dilution), anti-Fasciclin III monoclonal antibody 7G10 ( Patel et al. 1987 ) (1:50 dilution), and rabbit anti-β-galactosidase serum (Roche, Basel, Switzerland) (1:150 dilution) preadsorbed against Drosophila embryos. Secondary antibodies (Jackson Immunoresearch, West Grove, Pennsylvania, United States) were goat anti-mouse IgG-Cy3 (1:1000 dilution) and goat anti-rabbit IgG-FITC (1:300 dilution). Samples were blocked in PHT buffer (Ca ++ /Mg ++ -free PBS containing 1% heat-inactivated normal goat serum and 0.3% Triton X-100) for 1 h or more and then incubated overnight at 4 °C with primary antibody diluted in PHT. Samples were washed with fresh PHT at least six times for 1 h at room temperature, incubated overnight at 4 °C with secondary antibody, and washed as before. Samples were mounted in 70% (v/v) glycerol or Vectashield (Vector Laboratories, Burlingame, California, United States) mounting medium and observed with a Bio-Rad confocal microscope (Bio-Rad, Hercules, California, United States). Cy3 and FITC channels were sequentially excited and captured for each specimen; a Z-series of optical sections was collected and merged to avoid loss of out-of-plane information due to tissue wrinkling. Supporting Information Accession numbers The GenBank ( http://www.ncbi.nlm.nih.gov/ ) accession numbers for the genes discussed in this paper are bsk (NM_164901), Jra (NM_165739), kay (NM_170427), lz (NM_078544), msn (NM_079940), and puc (NM_079549).	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC479041.xml
545948	Critical evaluation of the JDO API for the persistence and portability requirements of complex biological databases	Background Complex biological database systems have become key computational tools used daily by scientists and researchers. Many of these systems must be capable of executing on multiple different hardware and software configurations and are also often made available to users via the Internet. We have used the Java Data Object (JDO) persistence technology to develop the database layer of such a system known as the SigPath information management system. SigPath is an example of a complex biological database that needs to store various types of information connected by many relationships. Results Using this system as an example, we perform a critical evaluation of current JDO technology; discuss the suitability of the JDO standard to achieve portability, scalability and performance. We show that JDO supports portability of the SigPath system from a relational database backend to an object database backend and achieves acceptable scalability. To answer the performance question, we have created the SigPath JDO application benchmark that we distribute under the Gnu General Public License. This benchmark can be used as an example of using JDO technology to create a complex biological database and makes it possible for vendors and users of the technology to evaluate the performance of other JDO implementations for similar applications. Conclusions The SigPath JDO benchmark and our discussion of JDO technology in the context of biological databases will be useful to bioinformaticians who design new complex biological databases and aim to create systems that can be ported easily to a variety of database backends.	Background Biological databases are key computational tools used daily by biologists. Such a large number of biological databases have been developed for biology that the Nucleic Acids Research Journal has published an annual database issue since 1996. From the point of view of the user, these resources are most useful when they are regularly updated and when they provide user-friendly ways to browse, search and view information. These user needs are generally recognized as important requirements by the designers and developers of biological databases. To cope with these requirements, bioinformaticians who develop the biological databases have typically responded by developing increasingly customized software to manage the data and the information (e.g., [ 1 - 4 ]). In doing so, and to facilitate the software development effort needed to create a biological database, bioinformaticians have used a variety of information technologies. These technologies range from the ones that make it possible to create dynamic web applications (e.g., Common Gateway Interface/CGI, Java Servlets, web application frameworks), to technologies needed to store the data and the information in a persistent manner (i.e., text files [ 5 , 6 ], relational databases [ 7 ], frame representation systems [ 8 - 10 ], object-oriented databases [ 11 ]). In this article, we report on our experience with the Java Data Objects persistence technology and take a critical view at the advantages and drawbacks of this emerging Java persistence standard for the development of advanced biological databases. We have ported the SigPath information management system (see below) to the JDO API and have defined an application-specific benchmark. We used this benchmark to evaluate the performance of two JDO implementations that target either a relational or an object database backend. This article summarizes the performance results that we obtained, announces the availability of the SigPath JDO benchmark (available under the GPL license), and identifies areas where the JDO API could be refined to facilitate portability and scalability of applications. Data persistence Biological databases are built with software that executes on computers. Most biological databases are of a size that could fit entirely in the central memory of modern computers. However, because computers may need to be shutdown for maintenance – or may crash inadvertently – data for a given database cannot be kept in computer memory for the life of a biological database. This problem is not specific to biological databases so that a variety of data persistence approaches and technologies are available. The key role of these technologies is to guarantee that data persists safely between the invocations of the programs that may modify the data. The pros and cons- of persistence technologies for biological databases Data can be stored in text files with limited structure and important information can be stored in unstructured text files expressed in English. Unstructured flat-files do not help perform large-scale analyses, structured queries or integrate data across multiple sources, all of which are important requirements for biological databases. Therefore, unstructured files are now widely recognized throughout the field as inadequate for the management of biological information. Highly structured data formats , such as ASN.1 [ 12 ] and more recently XML, are a more favored alternative. They can support structured queries, large scale analyses and data federation. Structured file formats, however, do not provide support for concurrent manipulation of the information by several users (e.g., several curators interacting with a submission tool to input new data about one protein in the database). As such, they are adequate for data exchange among systems, but not for concurrent access. Since the file format offers no support for synchronization, locking or complex domain-dependent data validation rules (for XML, XML Schemas are limited to simple validation rules), using structured data formats for biological information storage forces system developers to implement these services explicitly as a layer between the business code and the data storage. For instance, since XML Schemas are not capable of validating XML data with respect to information outside of the scope of the file being validated (such as data in other files or in a database), developers must implement custom validation code. XML Schema focus on syntactic validation, while most applications require semantic validation [ 13 ]. Database management systems (DBMS) have been historically developed to abstract the services (such as synchronization, business domain constraints) needed by systems that need to support large number of users accessing a shared storage of data. A few types of DBMS exist that differ in the way they represent data. Relational DBMS represent data as tables that contain rows and columns of various types, while Object DBMS support the concept of object classes and object instances directly. Relational DBMS such as Postgres, MySQL or Oracle have been used to store biological information in many laboratories, including ours [ 14 , 15 ]. A short introduction to using RDBMS for biological information storage was recently offered in [ 16 ]. Briefly, complex relationships among elements of information are stored in relational databases by expressing relations among records in several tables. The technology is useful for a variety of biological databases, where the mapping between the biological data and the relational data model is simple. However, the technology has two major drawbacks for advanced biological databases. The first problem is because of a mismatch between the object-oriented programming style and the relational data model. Advanced biological databases often require programs that manipulate tens or hundreds of object classes. Data in the instances of these classes needs to be made persistent, and this requires writing mapping code. The mapping code takes a graph of objects and transfers the data in this graph into records in the various tables of the relational database. Mapping code needs to be developed for the reciprocal operation, from the relational records to the object instance graph. Depending on the complexity of the relationships among objects in the graph, the development of the mapping code may represent a significant part of the code developed for the overall database. Object DBMS such as O2 [ 17 ] and FastObjects have been developed to eliminate the need to write mapping code, and to store objects directly in native form in the database. This approach was reported to offer substantial performance improvements and reduced development and maintenance costs for data organized in an object graph with complex relationships. Java Data Objects Technology The Java Data Objects Technology (JDO) is a Java application programming interface (API). This API was developed as a Java Specification Request [ 18 ] to offer: "a standard way to store Java objects persistently in transactional data stores..., a standard way to treat relational database data as Java objects, and a standard way to define transactional semantics associated with those objects." JDO appears as an attractive technology for the development of biological databases for the following main reasons: 1. It is designed to offer portability across a wide range of transactional stores or database backends, from open-source relational databases to native object oriented databases. 2. It transparently handles object persistence when relational or object persistence backends are used (the developer only manipulates objects and classes and does not need to write mapping code). 3. JDO also handles persistence transparently for object oriented databases, where mapping code is not needed. 4. It is a Java technology that integrates seamlessly with web application servers (e.g., Tomcat, JBoss, etc.) often used to create the web front-ends of a biological database. A critical evaluation of the JDO technology Given the stated advantages of the technology we decided to carry out a critical evaluation of JDO to determine if the technology can routinely be used for the development of advanced biological databases. Our evaluation focused on the following questions: Portability: Is JDO a mature API that can guarantee portability of the application across database backends? Performance: If portability is achieved, how do relational and pure object oriented backends compare in term of performance? Biological database specific requirements: Do complex biological databases have specific requirements that JDO 1.0.1 does not address? To answer these questions, we have ported a biological information management system (the SigPath system, see below) to the JDO 1.0.1 API. (The SigPath system was originally implemented with the ODMG API [ 19 ]). In the first step of the port, we compiled the new code with the FastObjects JDO implementation [ 20 ] FastObjects JDO is an implementation of the JDO API that connects to the native FastObjects object database. In a second step, we have adapted the existing code to support exchanging the JDO implementation and database backend between the FastObjects implementation and the Solarmetric Kodo implementation of JDO [ 21 ]. Kodo is an implementation of JDO 1.0.1 that connects to a variety of relational database backends. The aim of the second development was to modify the code to make it possible to switch from FastObjects JDO to Kodo JDO by changing a configuration property, and then simply recompiling. Our aim was to create a code-base that was fully portable from a relational database backend to an object-oriented database backend to address the portability question. The SigPath Information Management System SigPath is an open-source project aimed to develop an Information Management System (IMS) to foster modeling and simulation of cell signaling pathways and networks (see the SigPath project [ 22 ]) [ 23 ]. The SigPath IMS appears to the end-user as a web application that provides search, browsing and visualization capabilities. The project home page provides tutorials that explain how the system is typically used. Most traditional biological databases focus on one type of database entry (e.g., gene, mRNA, protein, protein motif, protein domain, etc.) and store information in database entries. This approach has been very useful to create detailed catalogs of biological parts and is a critical and essential element of the bioinformatics resources that support modern biological research. However, certain integrative studies, such as systems biology and modeling and simulation of biochemical pathways call for databases that integrate several types of information. The SigPath IMS is an example of an advanced biological database that encodes information through a number of information types and a set of relationships among them. Figure 1 illustrates how SigPath encodes information about a biochemical reaction: the reaction is represented as a graph of object instances. Introduction to the SigPath ontology/database schema A fragment of the SigPath ontology is given on Figure 2 as a UML diagram. The description of the complete set of persistent classes used in SigPath is given on the project web site ([ 22 ], see the "for developers" tab). The SigPath system supports several types of biological information, ranging from information to represent small molecules and proteins to the interactions between these molecules. The main information types supported by SigPath are listed on Table 1 . In SigPath, information is represented in an object-oriented manner, with information types often associated with classes. The SigPath object-oriented database schema was adapted from the EcoCyc ontology [ 9 ]. Several classes presented on Table 1 have an equivalent in the EcoCyc ontology. In the rest of this article, we will use the terms ontology and JDO database schema indistinctively, as they represent very similar concepts: a class in the object-oriented schema of SigPath is equivalent to a frame in the EcoCyc ontology, and an attribute of an object class is similar to the slot of a frame. This multiplicity of SigPath information types and the variety of relationships among them makes it important to clearly define what type of information can be represented by the system (i.e., the set of object graphs that could potentially be created and stored in the database). This information is formalized in the SigPath ontology. This ontology is implemented in a JDO database schema. (In the SigPath system, the set of allowed object graphs may be further reduced by adding semantic constraints to the validation mechanism used during information submission). The JDO schema consists of the set of SigPath Java classes that are persistent and of meta-data about these classes. Meta-data is expressed in JDO files and provides information about the classes that cannot be expressed directly in the Java language, for instance, type of the elements for the collection field of the persistent classes. Figure 5 shows a small JDO file and illustrates the type of information that it provides. A thorough presentation of the structure of JDO files is given in [ 24 ], vendor-specific extensions are documented in each JDO implementation. The SigPath code base has specific characteristic that make it a useful resource for evaluating JDO technology: • SigPath is an open-source project released under the GPL, so that the benchmark code is freely available for others to study, reproduce our results, or extend the benchmark to other JDO implementations or database backends. • SigPath is both a web-based application and a batch-oriented application. • The SigPath code-base includes unit tests [ 25 ] that help verify that the application behaves correctly against two different database backends. • The SigPath system provides varied use cases that exercise different behaviors of the database backend and JDO implementation (see use cases below). In the next section, we present the methods that we used to evaluate JDO technology for the creation of advanced biological databases. Results This section describes the results of the SigPath JDO benchmark and addresses the portability and performance questions described in the introduction. SigPath: porting from one JDO implementation to another We modified the FastObjects JDO version of SigPath to compile indifferently with the FastObjects and Kodo implementations of JDO. The modifications that we had to make to the project were (i) modifications to the JDO file, (ii) modifications of the code base and (iii) modification of the code base and application data. Modifications to the JDO file The Kodo enhancer tool performs stricter semantic validations on the JDO files than the FastObjects enhancer. Modifications needed to pass the validation tests were: 1. Added persistence-capable-superclass attribute to classes that have a persistence capable superclass. This attribute is optional for FastObjects, which uses Java reflection by default, but is strictly required by the Kodo implementation (in agreement with the JDO specification). 2. Removed all interfaces from the JDO file. Enhancing with Kodo failed when interfaces were listed in this file. Since FastObjects requires interfaces to be listed as persistent classes, the SigPath build script conditionally includes such statements in the JDO file when FastObjects is configured. The JDO specification does not mention interfaces, so that the behaviour of JDO implementation is left undefined. 3. (As a result of 2.) Replaced references to interfaces with references to implementation (e.g., replaced Protein by ProteinImpl) throughout the JDO file. 4. Added collection element types to all persistent collections. FastObjects requires the type to be specified when the collection is used in a query. Kodo requires the type to be defined for each collection, otherwise Kodo will try to serialize the collection and store it as a binary object. If the persistent class is not serializable, this mechanism will fail. Therefore, for this benchmark, we explicitly defined the collection types for each collection. 5. Removed field definitions from sub-classes when they refer to fields of a super-class. (e.g., the field "reactions" in Model was specified twice in the Model sub-class and in the Pathway super-class). Removing these duplicate declarations is consistent with the JDO specification. Furthermore, the Kodo enhancer expects classes to be listed in the JDO file in a specific order. The enhancer fails if a class appears in the JDO file before another class that the first class references. Therefore, we reordered the class definitions in the JDO file. (We verified that this is no longer an issue with version 3.0 of Kodo, but keep this description as other JDO implementations may share the same limitation). Finally, we added Kodo extensions to the JDO file to create indexes on the tables that were used extensively in queries. All changes to the JDO file were consistent with the JDO specification. Index tuning was performed by running the boot and test part of the benchmark and the small molecule import with various indexes choices. Modifications to the code base We modified the code base to work-around a limitation of the Kodo implementation. With Kodo, instances of classes that contain java.lang.Object fields are made persistent with the object field stored as a BLOB in the database. Storing objects as BLOBs puts strong limitations on their use. For instance, it is impractical to query for these objects by their fields (e.g., querying directly for a User instance by the id of the user is not possible if the instance is stored as a BLOB). Storing such fields as BLOBS was therefore not acceptable for certain types of persistence objects, and we implemented the work-around shown on Figure 3 . Another code modification was required to work-around a problem with the database backend that did not handle appropriately empty strings (“”). The database backend used for this benchmark stored empty strings as null. Reading these strings back from the database resulted in null being obtained from Kodo instead of empty strings. This resulted in several unexpected NullPointerException being thrown during the JUnit tests. Figure 4 illustrates the approach that we used to work-around this problem. Modification to the code base and benchmark/application data Finally, we had to modify the code base to put a limit on the length of long strings. Using a relational database backends imposes to define the maximum length of each string attribute defined in the persistent classes of the application. For instance, a limit must be set on the name attribute of the SigPathEntityImpl shown on Figure 2 . We initially used the default maximum length for all fields and found that certain fields could be longer than this limit when running the test and the benchmark. For instance, description fields of ProteinImpl are imported into SigPath from the DE line of SwissProt and TrEMBL entries. Some entries have long descriptions (that can exceed 1,000 characters). To test the impact of this limit on the code of the application, we arbitrarily choose to use a maximum length of 1,000 characters. We excluded from the benchmark input data proteins and small molecules that had aliases or descriptions longer than 1,000 characters, and other entries that would exceed any String field limit. This was done to make sure that the same input data was used for both the FastObjects and the JDO relational benchmarks. Performance measurements A brief summary of the performance measurements obtained with the SigPath benchmark is given in Table 3 . The table presents time measurements for each use case of the benchmark. The measurements are listed both for the FastObjects JDO implementation (columns marked FO) and for the Kodo implementation. Columns marked %FO/KODO indicate the percentage of the time running the benchmark with FastObjects takes compared to running the benchmark with Kodo. The last column of the table FO/KODO CV indicates the coefficient of variation of the total time across four independent measurements. Small values of CV (1–5%) indicate consistency between the four measurements. However, some use cases showed higher variations (10,11,12,36%), so we report as well the minimum value of the four time measurements for both FO and KODO (in columns marked Min). The raw data used for the calculation of these performance measures is provided in the supplementary material and on the SigPath JDO benchmark pages. These pages also provide the logs from which the raw data has been collected. Discussion Portability Our port of SigPath confirms that JDO greatly facilitates the porting of a bioinformatics application from one database backend to another. However, we report here several modifications that we had to make to the SigPath system to achieve this level of portability. This suggests that there is a need to develop JDO compliance tests that could be used to test that a specific implementation of a JDO-aware database is really compliant with the standard. This test suite would validate that JDO enhancers accept correct JDO files and correctly reject JDO files that break the specification. The differences in the interpretation of JDO files that we noticed between FastObjects and Kodo (see Results section) practically limit the portability of JDO applications. This article has presented techniques that can be used to work around these limitations until a JDO compliance test is developed and used. We note that the work arounds that we described may be specific to the two JDO implementations that we tested, and that other work arounds may be needed to achieve portability with other JDO compliant backends. Surprisingly, we found that an outstanding portability problem is in the way the different JDO back-ends store long strings of characters. While the FastObjects backend put no limitation of the length of long strings, the relational back-end used with Kodo limited the length of long strings to 4,000 characters. This limit had to be chosen and set for each persistent string field used in the application (when the default value was not appropriate). Although 4,000 characters may appear a large limit, it is likely to be reached in bioinformatics application either with textual or with sequence data. When this happens, the application will have to be re-engineered to work around the fixed limit. A work-around could be to use a data type that does not have a length limitation, but these data types also have other limitations (for instance, usually indexes cannot be used on those fields). Whichever solution is chosen, this issue must be considered early during the design of the application. It would be useful if the JDO standard offered a mechanism for the application developers to specify which string length their application requires to function properly with JDO backends. Each enhancer could then check that the application is requesting a maximum string length that is compatible with the database backend and fail early if it does not. (As of now, these types of error will most likely be detected when testing the application.) Performance The SigPath benchmark provides precise measurements of the performance of one biological database application against two JDO compliant database backends. The measurements were performed on use cases that are typical of the activities needed to develop the code of SigPath and to deploy a production SigPath system. (Table 2 . indicates which use cases belong to our software development process and which belong to administrative and curation tasks that we need to carry out to prepare a new release of SigPath). As shown in Table 3 , performance varies widely with the type of use case, but is overall significantly better with the object database backend. Use cases that perform batch loading of protein information into the database benefited the most from using the native object database FastObjects backend (with loading of data sometimes completed five times faster than with Kodo and a commercial relational backend). An exception to this trend is the SM Import use case, which shows only a 3% performance difference. This use case reads an XML file and loads small molecules into the database. To do so, it checks for each molecule that the accession code of the new molecule does not already exist in the database (this is an error condition that would interrupt the import). Since the database does not contain small molecules, the query used to perform this check returns an empty set for each molecule of the import. It appears that this specific operation is slower with the object-oriented backend that we have used for the benchmark. The last column of Table 3 indicates the coefficient of variation (CV) of the individual measurements (among four independent executions). The CV values indicate that the performance of certain steps vary significantly from execution to execution. These differences are likely to be caused by the caching behavior of the database server and of the operating system. Caching can occur because we have not restarted the database server between the benchmark runs, or rebooted the machines. These differences may also be caused to a lesser extent by variations in what operating processes were active and the amount of IO wait at the time that the specific use case was executed. We have tried to reduce such causes of variability (see methods) but have not attempted to eliminate them completely (e.g., setup an isolated database server and disable all interactive use of the server). Our rationale is that such variability, including caching, is representative of a typical production system. Given the CV, the average execution time may not be an accurate representation for some use cases, so we report also the minimum execution time across the four independent executions of the benchmark. The benchmark provides an indication of how well an object-oriented database system performs compared to a relational database backend for the SigPath use cases. A known limitation of benchmarks is that the performance measure that they provide are specific to the application tested, and may not generalize well to other use cases. Also, the SigPath benchmark does not cover multithreaded/multiclient operations. Results may vary depending on the chosen locking strategy and the number of clients/threads running parallel. Given these caveats, however, this benchmark indicates that, for most of the SigPath use cases, the performance of the SigPath system is significantly improved when using a native object database system. Particularities of the SigPath benchmark that may correlate with this result are (i) the complexity of the database schema (75 persistent classes) and (ii) the number of connections that exist among instances of these various classes. Finally, these results and our distribution of the SigPath benchmark source code can help vendors diagnose performance problems with their implementation of the JDO implementation, and provide users with an objective measure of the performance a given JDO implementation, for similar types of applications. Biological database specific requirements During our evaluation of the JDO technology, we have noted that two common requirements of advanced biological databases are currently not being addressed by JDO. Support for interfaces When designing a biological database schema, it is often useful to express that one class shares the properties of two or more classes. In a programming language such as C++ this can be represented as multiple inheritance (one class inherits from two parents) while in a programming language such as Java, this concept is represented with interfaces (one class implements two interfaces). In the context of JDO, consider the class diagram shown on Figure 6 . The diagram illustrates one way to represent the phosphorylated forms of protein and small molecules. On this diagram, one has represented a "Phosphorylated" interface which is implemented by PhosphoProtein and PhosphoSmallMolecule. While this way to represent biological information is useful, JDO does currently not specify the handling of interfaces, so that the design shown on Figure 6 can not be implemented with JDO in a portable way. (This design would work with FastObjects, but not with Kodo.) The JDO specification should clarify if interfaces must be supported by for a JDO implementation to be compliant with the standard. Support for large number of objects Biological databases often need to manage large number of objects (e.g., large number of proteins, small molecules, etc.). For instance, SigPath stores information about several hundred of thousands of proteins. We found that JDO 1.0.1 lacks some features that would facilitate writing scalable applications. An example is that the JDO standard does not provide a scalable way to determine the number of persistent instances of a given class. The JDO compliant way to accomplish this operation is to obtain a reference to a collection of instances of this class (using a JDO Extent), and to call the size() method on this collection. Since the collection must first be obtained from the database server before the size() method can be invoked, this procedure takes a time proportional to the number of instances of this class. Most database backends store the number of instances of a certain class in the database and can determine this information in a constant time, so a standard way to obtain this information from a JDO implementation would be very helpful (SigPath can use either a pure JDO extent sizing method, or a vendor-specific method through an extension mechanism implemented in the source code, so that performance can be compared). A second example is that JDO 1.0.1 does not provide support for queries that return large result sets. Under standard JDO 1.0.1 behavior, traversing a persistent collection (by accessing each element of the collection in turn) brings the entire contents of that collection into memory. This behavior is appropriate for small result sets. However, there are cases where the complete set of instances returned by a query cannot be processed within a single transaction. This occurs for instance when all the results returned by a query do not fit in the fixed memory limit allocated to the Java Virtual Machine. In such cases, it may be necessary to obtain the result of a query in chunks of a certain number of records/instances (for instance 1,000 or 10,000 instances at a time), and process them in independent transactions. Upon transaction commit, memory associated with a chunk is released and can be used to process the next chunk. Implementing this type of scalable processing in an efficient manner usually requires making modifications both in the persistent class of elements in the result set and in the query filter. The class of the elements in the result set can be modified to add an instance identifier that can be used both to sort the instances and to select only those within the current processing chunk. The query filter can be modified to add a clause that selects only instances of the next chunk, based on the identifier introduced in each element. An alternative is to provide an API call to notify the JDO implementation that instances which have been processed can be evicted from memory. Since several vendors already have their own extensions to provide scalability feature, it would be useful for JDO to support such features through a standard API. Conclusions Here, we have shown that it is possible to develop a bioinformatics database that can be reconfigured automatically and recompiled to run either against a relational database backend or against an object database backend. The key advantage of this added flexibility is that the bioinformatics database becomes portable with respect to the database backend. This has important implications for the development of open-source bioinformatics databases. In such projects, usually more than one laboratory contributes to developing the software of a specific biological database. Therefore, it is useful if each laboratory can choose a database backend for development and deployment, yet contribute to the project in a shared code base. The Java Data Objects standard offers the productivity gains of transparent object persistence, and a fine-grained object persistence model useful to represent many biological concepts. We discussed why JDO can appear as an attractive option for the development of advanced biological databases and the type of problems that we encountered when implementing and deploying a biological database against two different JDO implementations. The future JDO standard (JDO 2.0) should address some of the issues that we discussed in this article (e.g., support for interfaces, or for large result sets). When JDO 2 implementations become available, we expect that JDO technology will have a significant impact on the design of high-performance biological databases that need to represent and manage complex biological information types. Methods Benchmark use cases To address the performance question, we have developed benchmark use cases. The benchmark use cases were designed to be representative of performance that one would observe when either (i) developing the software of the SigPath system or (ii) preparing a new release of the SigPath IMS (includes loading the database with information from other databases). Our benchmark thus considers both the development and the production stages of the life-cycle of the application. The use cases, or benchmark steps and a summary of their purpose in the context of the SigPath project are listed on Table 2 . The boot and test steps make it possible for the SigPath developers to create a sample database and test that important functionalities of the application are working satisfactorily. boot – The boot step compiles the sources of the project, enhances the JDO persistent classes (a program, called a JDO enhancer, transforms Java class files into persistent classes and allow them to interact with the JDO implementation), creates an empty database and imports information into the database. Importing this information involves parsing an XML file that contains the information, validating this file against the SigPath information exchange XML Schema, validating against additional semantic rules that cannot be expressed with XML Schemas (database lookups are used during this step to connect new instances to instances previously submitted in the database, if needed), and saving new persistent instances to the database. The boot sample data is designed to contain at least one instance of each type of information that can be stored in the SigPath IMS. test – The test step runs JUnit tests against the data that was imported during the boot step. The JUnit tests assert that information stored in the database corresponds to the information in the boot XML file. For instance, the tests check that the number of persistent instances matches the number found in the boot import file, but also that specific elements of information have been saved accurately. Furthermore, the tests assert various semantic properties of the application and database access code, running queries against the database, navigating through objects, creating new persistent instances or deleting them, etc. The complete set of operations performed in the test is fully described in the source code for the JUnit tests (see edu.mssm.crover.sigpath.test package, and specifically the class MasterTest). small molecule import – This step creates SmallMoleculeImpl persistent instances (implementation of the SmallMolecule interface shown on Figure 2 ). The data used to create these molecules is a modified form of the NCI open database. Only small molecules that have a name, description, aliases and SMILES representation are imported from NCI Open (the total number of molecules read is 237,771, and the total number of molecules loaded into the database is 45,229). These data are imported and stored in the attributes of SmallMoleculeImpl (most fields: name, description and aliases are inherited from SigPathEntityImpl). protein import – This series of steps creates ProteinImpl persistent instances (implementation of the Protein interface, shown on Figure 2 ). The data to load these proteins is obtained from a simplified XML format created from SwissProt and TrEMBL data files with SwissKnife [ 26 ]. The exact list of files imported is given on Table 2 . full text indexer – The SigPath system offers users the ability to search entities by keywords. This step builds an inverted full text index [ 27 ] that is used by the web application to accelerate keyword-based queries. An inverted full text index links each keyword that occurs in text strings of a SigPath entity (e.g., name, description, aliases) to the SigPathEntity instance that contains the keyword. This step creates 465,679 Keyword instances that link to a total of 345,133 SigPath entities (small molecules or proteins). XML import – This step is similar to the loading of SigPath information in the boot target, but loads information obtained from the online version of SigPath (this benchmark used the information as of October 14 th 2003). For this benchmark, XML import instantiates 14 SmallMoleculeImpl, 8 ProteinImpl, 121 ComplexImpl, 77 modified chemicals (SmallMoleculeImpl or ProteinImpl), 92 ConcentrationMeasurement, 165 ReactionImpl, 75 EnzymaticReactionImpl, 23 Model, 3 Pathway and 27 PendingReviews. Benchmark procedure The benchmarks were run as described on the SigPath Project web site ([??] see the "JDO benchmark" tab). Each benchmark (FastObjects or Solarmetric Kodo with a relational database) was run on a two Xeon 3GHz processor machine with hyper-threading on and 6 Gb of memory. The machine was running Red Hat Advanced Server Linux 2.4.21-4.0.1.ELsmp, and was used both as database server and database client (to minimize the impact of the network on performances). No significant other processes were running on the benchmark machine while the tests were executed. We benchmarked FastObjects t7 server version 9.0.7.185 and Kodo JDO version 2.5.3. Each benchmark was run four times to average the effect of variability in the computational environment that may not have been controlled by our benchmark procedure. The results report the coefficient of variations (mean divided by the standard deviation) of the total running time for each use case and this helps point out cases when the computational environment had an effect on measured times. We believe that these variations are common in a production environment and report the average total running time as well as the minimum total running time for each use case. Authors' contributions Marko Srdanovic and US implemented significant components of the FastObjects and Kodo JDO ports. Marko Srdanovic collected benchmark data at WMC and US collected similar data at FastObjects. Michael Schwieger and FC designed the study and contributed to the JDO ports. FC drafted the manuscript. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545948.xml
555597	Modeling emergency department visit patterns for infectious disease complaints: results and application to disease surveillance	Background Concern over bio-terrorism has led to recognition that traditional public health surveillance for specific conditions is unlikely to provide timely indication of some disease outbreaks, either naturally occurring or induced by a bioweapon. In non-traditional surveillance, the use of health care resources are monitored in "near real" time for the first signs of an outbreak, such as increases in emergency department (ED) visits for respiratory, gastrointestinal or neurological chief complaints (CC). Methods We collected ED CCs from 2/1/94 – 5/31/02 as a training set. A first-order model was developed for each of seven CC categories by accounting for long-term, day-of-week, and seasonal effects. We assessed predictive performance on subsequent data from 6/1/02 – 5/31/03, compared CC counts to predictions and confidence limits, and identified anomalies (simulated and real). Results Each CC category exhibited significant day-of-week differences. For most categories, counts peaked on Monday. There were seasonal cycles in both respiratory and undifferentiated infection complaints and the season-to-season variability in peak date was summarized using a hierarchical model. For example, the average peak date for respiratory complaints was January 22, with a season-to-season standard deviation of 12 days. This season-to-season variation makes it challenging to predict respiratory CCs so we focused our effort and discussion on prediction performance for this difficult category. Total ED visits increased over the study period by 4%, but respiratory complaints decreased by roughly 20%, illustrating that long-term averages in the data set need not reflect future behavior in data subsets. Conclusion We found that ED CCs provided timely indicators for outbreaks. Our approach led to successful identification of a respiratory outbreak one-to-two weeks in advance of reports from the state-wide sentinel flu surveillance and of a reported increase in positive laboratory test results.	Background Traditional public health surveillance for specific conditions is unlikely to quickly identify a disease outbreak. Emergency department (ED) data appears to have the potential for more timely disease surveillance [ 1 - 6 ]. In non-traditional surveillance, signs of an outbreak might include an increase in ED visits for respiratory, gastrointestinal or neurologic chief complaints (CC). Crucial to any surveillance is an understanding of normal patterns in the data. Utilization patterns in the ED are thought to be difficult to predict, due to large variability that arises in part because EDs are required to medically evaluate and stabilize everyone who requests care. Therefore, ED visit rates cannot be controlled by insurers, institutions or policies. Many "drop in" surveillance systems have been hampered by a lack of knowledge of baseline activity [ 7 ]. Other systems have used short-term moving averages of the recent past to predict current usage [ 8 ], but this does not provide optimal performance when day-of-the-week or seasonal effects are smoothed away by the averaging. Some "active" ED surveillance systems provide on-going data collection. The EMERGEncy ID NET [ 9 ] and RSVP [ 10 ] function as sentinel surveillance systems where data from a small sample used may not represent the overall occurrence of disease. If data collection is inconsistent, it does not provide reliable information about syndrome incidence. This paper reviews our experience with an operational near-real-time surveillance system, the Bio-Surveillance Analysis, Feedback, Evaluation and Response (B-SAFER) system. B-SAFER is the result of collaboration between the Los Alamos National Laboratory, the University of New Mexico Health Sciences Center, and the New Mexico Department of Health. Medical surveillance systems such as B-SAFER require considerable expertise in computer science and systems integration for their design and architecture, to comply with security and privacy issues, and to ensure timely flow of information [ 11 , 12 ]. These systems also require medical and epidemiological expertise to identify appropriate items to monitor for anomalous events and to understand their significance. Methods Setting This observational study uses data from the Emergency Center of the University Hospital, Albuquerque NM (UH), a tertiary-care county-university health sciences center. The Emergency Center includes pediatric and adult emergency departments, an urgent care center, a trauma center and an observation unit. There are roughly 200 patient visits per day, or 73,000 per year, representing 36 % of Emergency Department visits in Albuquerque. This study was approved by the Institutional Review Boards of the University of New Mexico Health Sciences Center and Los Alamos National Laboratory. Data stream The data is from the computerized ED patient tracking system in place since 1994. Data in the system includes: date and time of arrival and discharge, age, sex, chief complaints, discharge diagnoses and disposition. CCs are recorded by the nurse at the time of triage and entered into the system by a clerk. The clerk may select from a drop-down menu of complaints or may enter the complaints as free text. The menu option is rarely used because clerks find free-text entry more flexible and convenient. We group daily CC counts into seven categories: respiratory, gastrointestinal (GI), undifferentiated infection (UDI), lymphatic, skin, neurological, and "other" (Table 1 ). The "other" category includes all visits except those in the first six categories. These grouping categories are also used by other surveillance programs, such as ESSENCE (Walter Reed Army Institute of Research) and the Real-time Outbreak Detection System ((RODS, University of Pittsburgh) [ 11 , 13 , 14 ]. Our grouping scheme is provided in Table 1 . Table 1 B-SAFER dictionary for matching chief complaints to body systems Respiratory Gastro-intestinal Neurologic Skin Lympatic UDI (undifferentiated infection) Breath Bronchiolitis Chest congestion Chest pain Cold Congested Congestion Cough Croup Flu Headache Laryngitis Pneumonia Respiratory Sinus Stuffy nose Throat Abdominal Pain Abdomen/back pain Abdomen pain Abdominal cramps Abdominal pain Blood in stool Diarrhoea Food poisoning Hepatitis Jaundice Stomach pain Vomit Nausea Non responsive Altered mental status Anxious Confusion Difficulty Talking Difficulty thinking Difficulty Walking Disoriented Drowsy Facial droop Facial weakness Hyper Loss of consciousness Mental Nervous Numbness Paralysis Seizure Slurred speech Sores Stroke Swallowing Syncope Thinking slow Tingling Trouble talking Trouble thinking Trouble walking Unresponsive Weak Abscess Abnormal Skin Blisters Bug Bites Cellulitis Chicken pox Dermatitis Insect bite Itching Pox Rash Skin redness Skin swelling Tick bite Arm pit Glands Lumps Lumps in neck Neck Nodes Red streaks Skin streaks Weak Achy Body aches Body sores Fatigue Fever Fussy Infection Tired To obtain this scheme, we reviewed a frequency table of all CCs which occurred at least 5 times over nine years and assigned relevant CCs to groups, as was done for example in [ 15 ] and [ 16 ]. Key words were selected to capture multiple chief complaints containing that word. For instance "breath" captures "shortness of breath", "trouble breathing", "I can't breathe", "can't catch breath", "breathing problems", etc. Common abbreviations were also included as key words. The groupings were then reviewed by the project medical advisory board which included epidemiologists, infectious disease, emergency and occupational health physicians. Each CC is assigned to a group when the first match was made between a word in the CC and a word in our CC dictionary. There were no examples of negative chief complaints such as "no cough", in our nine-year database, so we did not develop a system for handling these. Any negative complaint would have been classified into the category of the key word. First order model The training set for model development was retrospective cohort data from 2/1/94 through 5/31/02. We used least squares regression in started log scale to fit trends, seasonal effects, and day-of-week effects. The "started log" is the logarithm of one plus the number of daily CCs. We add one before taking the logarithm to avoid problems with taking the logarithm of zero counts. The started log scale results in more symmetrically distributed forecast errors with variance that is much less dependent on the mean count. Results are back-transformed to natural scale for display. Error bars behave as desired (widening when the CC count increases) and skewing is commensurate with the magnitudes of the baseline values. A model that incorporates the above effects is S (d) = [Σ i c i × I i (d) ] + [ c 8 + c 9 × d ] + [ c 10 × cos (2π d / 365.25) + c 11 × sin (2π d / 365.25)] where a) S ( d ) denotes the started log of the number of chief complaints) for day d , where "Day 1" is taken as February 1, 1994, b) [Σ i c i × I i (d) ] captures the day-of-the-week effect, where the sum is over the indices i = 1 to 7 and I i (d) denotes the indicator function for day d , i.e., I i (d) equals 1 when day d is the i- th day of the week and equals zero otherwise, and the seven model coefficients { c i } are constrained to sum to zero, c) [ c 8 + c 9 × d ] captures a long term linear effect, d) [ c 10 × cos (2π d / 365.25) + c 11 × sin (2π d / 365.25)] captures the seasonal component, where the average number of days per year is 365.25, with the model coefficients c 10 and c 11 dictating the time and amplitude of the seasonal effect. This model arises from transforming the counts to started log scale and adding day-of-the-week effects to the regressor variables in a cyclical regression model [ 16 , 17 ]. Application of the model allows for coefficients to be set to zero when the corresponding constituent effects are not statistically apparent. For example, the coefficient c 9 is zero for complaint categories that do not exhibit linear long term trends, and the coefficients c 10 and c 11 are zero for categories which do not exhibit seasonality. A plot of the predicted respiratory complaints and corresponding upper confidence limits from this model is given in Figure 1 , illustrating how the constituent effects interact to yield baseline predictions. The predictions were obtained using ordinary least squares for parameter estimation and then back transforming from started logs to natural scale. Figure 1 Predicted Respiratory Complaints. The predicted respiratory complaints and corresponding upper confidence limits illustrates seasonality and day-of-week effects. Evaluation of goodness of fit As with all statistical models, it is important to assess goodness of fit. A careful residual analysis reveals trends in the forecast errors, the most important of which follow from the one-size-fits-all character of the model. That is, the first order model postulates that complaint activity peaks with the same magnitude and at exactly the same time of year from season to season. Such postulated behavior is only approximately true, and the season-to-season differences lead to the trends in residuals from the model. For example, if a peak of respiratory complaints occurs later than average in the year, then the baseline will initially over-predict (in anticipation of an average peak time) and then under-predict (when the season's peak actually occurs). Similarly, if the amplitude of a season's peak is higher or lower than the historical average amplitude, predictions will be consistently too low or too high near the peak. We return to this subject in the section on hierarchical modeling. Near real time monitoring: Page's test By comparing CC counts to predictions and confidence limits, anomalies can be identified [ 18 , 19 ]. Extra counts could arrive all on one day, or appear as a gradual increase starting at some particular time, or arrive sporadically, persist at a constant level for a fixed duration, etc. The best statistical test for detecting extra counts depends on the pattern of extra counts, so there cannot be a single best test for detecting all possible anomalous patterns. A particular test, based on Page's statistic, is optimal for detecting a constant excess above baseline when the start time and duration of the excess is unknown [ 20 ]. This test also has competitive power compared to other sequential tests to detect other anomalous patterns. For these reasons, Page's statistic is widely used in statistical process control and has been proposed in the context of disease surveillance [ 21 ]. We recommend Page's statistic unless specific anomalous patterns are suspected, in which case a specialized test could be developed. Page's statistic is a type of cumulative sum, which we denote as P ( d) . On each day d , the forecast error ε d between the started log of the observed number of complaints and the started log of the baseline prediction is computed for each complaint category. The standard deviation s d of ε d is computed as well. Then Page's statistic is calculated for day d as P ( d ) = maximum of 0 and [ P ( d -1) + ε d / s d - 1/2]. If P ( d ) becomes too large, then the observed complaint levels are significantly greater than the baseline predictions and complaint levels are deemed anomalously high. Here, the phrase "too large" is formally defined in terms of the desired false positive rate for monitoring, and the threshold value for P ( d ) is calibrated using empirical data to account for model prediction errors. Comparison to other data streams A comprehensive comparison to other data streams is beyond our scope. Briefly, we compared our respiratory CC counts to existing influenza data for the 2002–2003 influenza season. We evaluated New Mexico (NM) Department of Health sentinel influenza surveillance data reported weekly by approximately 20 clinics. When the week-long monitoring period is combined with the time needed for compilation and dissemination, notification of an outbreak early in one week is often not formally received until two weeks after it occurred. We obtained virology laboratory data from routine clinical and surveillance testing of respiratory specimens reported by three laboratories that conduct at least 75% of the clinical virology testing for NM. Such data streams have their own timeliness issues, due in part to culturing of samples. Results ED data from 2/1/94 – 5/31/02 is used as training data for least squares fitting to establish control limits; data from 6/1/02 – 53/31/03 is then used in near real time surveillance. Approximately, 17% of the complaints fall into the respiratory category, 10% gastrointestinal, 6% undifferentiated infection, 3% skin, 3% neurological, 1% lymphatic and 60% "other." Day-of-week effects For all systemic complaint categories there are day-of-week differences. See Figure 2 . For five of the seven categories, there are more visits on Monday than on any other day; UDI and skin peak on Sunday. Weekly minimums occur later in the week: on Thursday for skin, Friday for GI and UDI, and Saturday for respiratory, neurologic and lymphatic. In some cases, there is a high-to-low trend as the week progresses. Figure 2 Day-of-week effects for each CC category. The average day-of-week effect with corresponding error bars for six CC categories. For respiratory and UDI complaints, there is an average difference of 7 cases per week between Monday and Friday, and the weekly differences conform to a bell-shaped statistical distribution. While day-of-week effects are statistically significant in all categories owing to the size of the data set, in some categories there are so few complaints that the difference is of no practical consequence. For example, in the lymphatic and neurologic categories, the average difference between the weekly peak (Monday) and weekly minimum (Saturday) is less than one case per day. Certain other daily effects may exist, e.g. holiday effects [ 22 ], but sample sizes for UH data are not large enough to detect them. Seasonal effects As is well known, there are annual cycles of respiratory complaints with peaks in January or February. Figure 3 demonstrates these cycles in our data for respiratory complaints; the cycles are similar but less pronounced for UDI complaints. Figure 3 Annual cycles in Respiratory Complaints. Annual cycles in respiratory complaints (by week) for the past three flu seasons, from 2000–1 through 2002–3. Long term trends Total ED visits increased over the study period by 4%, in part reflecting the population increase of about 1.5 % per year for the metropolitan area. Rates for most of the complaint categories have changed over the eight years that data has been collected. Respiratory complaints show a decrease of roughly 20% over the monitoring period (Figure 4 ), illustrating that long term averages need not reflect current or future behavior. Skin-related complaints also show a slight decrease, while increases are observed in nearly all other categories. Only lymphatic complaints do not appear to change over the monitoring period. Had there been a complaint category where a nonlinear trend were clearly present, either the c 9 component would have been modified periodically, or a nonlinear model would have been used. Figure 4 Average daily number of respiratory complaints by calendar year. The average daily number of respiratory CCs decreases over the training data. Surveillance Patterns observed in the Test Year We focus on respiratory CCs because they have the strongest season-to-season variation, which makes them the most challenging to predict, and because respiratory is thought to be one of the most important bioterrorist categories. Overlaying the data streams on the baseline plot in real time allows for visual inspection of the results, similar to that for a standard control chart. Figure 5a shows the daily counts, the baseline prediction, and the upper control limit (for a 2.5% false alarm rate) for the respiratory category. Figure 5b shows the scaled forecast errors (in the started log scale) for the prospective data. Both figures reveal a later-than-average flu season, as does Figure 5c , showing large values of Page's statistic P (d) . These plots illustrate how a misfit to the one-size-fits-all model can produce systematic trends in surveillance data. This subject is revisited in the section on hierarchical modeling. Figure 5 Results on validation data for Respiratory Complaints. Prospective data (June 1, 2002 through May 31, 2003 (a) Daily, predicted, and upper control limit for respiratory counts ; (b) Scaled forecast errors for respiratory counts; (c) Page's statistic applied to the same forecast errors. A control value of 3.3 bfor Page's statistic results in an approximate theoretical 2.5% false alarm rate when forecast errors are Gaussian. The peak in respiratory CCs and the elevated P(d) preceded a rise in reports from the state-wide influenza sentinel surveillance system. A similar pattern, delayed by several weeks, was found in the rise of requests for laboratory tests for influenza. ED CCs also preceded New Mexico reporting of deaths from pneumonia and influenza. We conclude that surveillance using the first order model is sufficiently sensitive to mild departures from baseline activity and that it can provide timely notification relative to traditional surveillance. Simulated outbreaks In a simulation study we injected K extra respiratory CC counts beginning at random days during the test year, with the simulated outbreak lasting from 1 to 10 days, from 2 to 10 days, and exactly 1 day. Generally, departures of approximately 3 or more standard deviations from the baseline model should be detected with high probability. The simulated per-day shift above the baseline prediction ranged from 1 to 5 standard deviations in our simulations, so testing one day at a time could fail to detect those outbreak having small per-day shifts. Also, because of the pattern in the residuals near each seasonal peak, we considered EWMA (exponentially weighted moving average, see the Discussion) as one way to modify the current forecast on the basis of errors in the recent past. In Table 2 we give the fraction of simulations (out of 1000) in which the Page statistic exceeded its threshold of 3.3 for the null model, baseline model, and for the same models modified by the EWMA procedure. For comparison to one-day-at-a-time testing, we also give the fraction of simulations in which the maximum forecast error that occurred during the outbreak exceeded its 2.5% false alarm rate threshold of 1.96. We see that Page's test outperforms the one-at-a-time test and that the EWMA modification does not improve anomaly detection because of its tendency to underestimate the size of multiple-day outbreaks. However, if we restrict attention to those outbreaks that last only one day, then one-at-a-time testing is better (for each of the models), as we would expect. Compare the baseline model results to the null model (which uses the average CC count in the training data to predict the test data) results to gauge the benefit of fitting the baseline model. Of course the null model is not acceptable regardless of its performance in this context because it ignores the trend (which causes the null model to be biased high for the respiratory CCs), day-of-week effects, and seasonality. Table 2 The fraction of simulations (out of 1000, so the 95% confidence limit is approximately ± 0.03) in which the Page statistic (or the one-at-a-time statistic) exceeded its 2.5% false alarm threshold during the simulated outbreak for the baseline model, the baseline model with residuals modified by EWMA, the null model, and the null model with residuals modified by EWMA. Outbreak duration Test Baseline Baseline + EWMA Null Null + EWMA 1–10 days Page 0.46 0.21 0.28 0.17 1–10 days One-at-a-time 0.37 0.31 0.17 0.31 >1 day Page 0.42 0.20 0.30 0.14 >1 day One-at-a-time 0.31 0.25 0.13 0.26 1 day Page 0.44 0.36 0.30 0.45 1 day One-at-a-time 0.71 0.70 0.47 0.68 Discussion Models Long-term trends can occur in surveillance data for multiple reasons. Changes may occur in: local resources (more or specialty EDs), access and reimbursement practices (facilities change which insurance plans with which they are associated, major shifts in insurers drives patients to other facilities), changes in the underlying population (shifts in population size or age), and changes in the local economy. Moving averages were not used because although they generate visually pleasing curves they smooth over day-of-week and seasonal effects that are important for developing baselines. Concerning model quality, one useful test is whether the forecast error variance in the testing data is approximately the same as that in the training data. Upon dividing the forecast errors in the test data by their standard deviations in the training data, the scaled forecast error variances range from 0.87 to 1.07 for the seven CC categories (ideally, these ratios should be near 1). Further, the fraction of scaled forecast errors that exceed 1.96 ranged from 0.0 to 0.033 (when the model holds and residuals are Gaussian, the portion of one-sided residuals exceeding 1.96σ is 2.5%). Thus, departures from stationarity in the time series are mild enough that the forecast errors show that future complaints can be reasonably well predicted using a single baseline model for each category. When monitoring complaint levels over multi-year time frames, it is necessary to periodically update baseline model coefficients in order to minimize the extrapolation in forecasting. One approach to choosing an update frequency is to do a planned update every year, but also monitor residuals for patterns, including shifting variance, that have not been observed previously to check whether additional updates are needed. Hierarchical modeling to capture season-to-season differences The first-order model is useful for routine monitoring. It has the obvious shortcoming, however, of describing each season in a one-size-fits-all fashion. As noted above in evaluation of the model's goodness-of-fit, forecast errors reflect modelling imperfections as well as random variability, limiting somewhat the sensitivity of surveillance to detect smaller anomalies. Improving on this situation requires more refined baselining. Hierarchical methods [ 23 ] can overcome the one-size-fits-all shortcoming, or, at a minimum, provide information that is valuable in assessing the quality of one-size-fits-all modelling assumptions. In the hierarchical approach, each season is allowed to have its own time of peak activity, its own seasonal duration, and its own peak magnitude. For practical purposes, the hierarchical model shares the global characteristics of the first order cyclical regression model. The seasonal component is modelled with a scalable Gaussian function, in contrast with the fixed-width sine and cosine harmonics previously. And the underlying baseline changes linearly within a season, as opposed to behaving linearly over a longer time period. Applying the hierarchical model to respiratory CC data illustrates the season-specific nature of chief complaints. On the average, our respiratory complaints peak on January 22, with a season-to-season standard deviation in the day of the peak of 12 days. The durations of individual seasons, defined in terms of the standard deviations for the Gaussian-shaped peaks, vary by factor of two over the monitoring period. And there is no apparent relation between the time that the peak occurs and the magnitude of the flu season. Use of hierarchical models for real time syndromic monitoring could be considered, but at a significant computational cost. In order to capture the peak time and magnitude of an ongoing season, the model must be updated on a frequent (e.g., weekly) basis, involving lengthy runs of Markov chain Monte Carlo software. Because the first order cyclical regression model fits the data sufficiently well to detect anomalies of interest, we have used the first order model for routine monitoring. A similar first order cyclical regression model is used by the Centers for Disease Control to monitor pneumonia and influenza related mortality data [ 24 ], also with success. Related efforts Influenza surveillance basing alerts on comparison to historical data were described by Irvine [ 25 ]. Daily counts were compared to historical averages and standard deviations. Their data demonstrated a peak in CCs during influenza season. Lazarus et. al. [ 26 ] use a generalized linear mixed model based on four years of data from ambulatory health encounters. They find that indicators for day-of-week, month, holiday effects as well as a secular trend term contribute significantly to their model fit. There may be ED data from other hospitals where month-to-month effects exist but are not part of a longer seasonal trend, but we don't see them in our data. Logistic regression [ 26 ] is useful for scaling over census tracts of different population sizes and, when complaint counts behave proportional to underlying census populations, is also useful in modeling overall complaint levels. Reis and Mandl [ 27 ] used CCs for their time series models (autoregressive integrated moving average, ARIMA, models) for total and respiratory visits. After fitting a day-of-week effect and a seasonal effect, there remained positive autocorrelation in the forecast errors, which they modelled using a particular time series model. In our CC data, there is negligible autocorrelation in the errors after fitting our model, except that due to the variation in when the seasonal peak occurs. For example, if a peak occurs early, then we observe a sequence of positive errors, which leads to positive autocorrelation of the type reported. For our CC data, the best-fitting ARIMA-type model applied to the residuals after fitting the trend, seasonality, and day-of-week effect was the EWMA (equivalent to a moving average fit to the first differences, also denoted ARIMA(0,1,1) for the particular autoregressive integrated moving average model that it corresponds to). Reis and Mandl [ 27 ] note that the ARIMA model adjusts to multi-day outbreaks and so it reduces the error on days 2, 3, ... of a multi-day outbreak. Therefore they suggest using both the original errors (containing serial correlation) and the ARIMA-model-adjusted errors in two monitoring schemes. The EWMA also adjusts to multi-day outbreaks and therefore suffers from signal loss if the outbreak persists for multiple days (the results in Table 2 illustrate this effect). Therefore, the Reis and Mandl [ 27 ] suggestion to monitor two residual series is relevant if we use EWMA or any approach (including the hierarchical model) that uses the very recent past (in addition to the trend, seasonality, and day-of-week effects) to modify the current forecast (leading to two or more forecast methods). Also, sequential tests were not used in [ 27 ] for their 7-day simulated outbreaks. Each simulated outbreak added simulated additional counts to the daily CC data. Forecasts were made (on the basis of a model that used the overall mean, the day-of-week means, and the trimmed day-of-year means) and were modified using the ARIMA-modeling of the residuals. If any single-day forecast error exceeded a threshold, then the simulated outbreak was said to be detected. Sequential tests are ideal for multi-day outbreaks so the performance (the false negative rate for a fixed false positive rate) of Page's statistic or a moving window such as in [ 28 ] or the scan statistic such as in [ 29 ] would be better than the performance of one-at-a-time tests in the case where all simulated outbreaks lasted 7 days. Reis et al. [ 28 ] applied several sliding detection windows, each of at most 7 days to ED visit daily counts in which simulated outbreaks (in the form of additional ED visits) lasting 3, 7, and 14 days were added to the real data in a simulation study. On the other hand, if each outbreak lasted only one day, then monitoring single-day errors would be optimal. In summary, we concur with [ 30 ] regarding the robustness and simplicity of Page's test. Alternatively, there are occasions when using a modest number of specific tests is effective as was done in [ 29 ]. Use of free text chief complaints Many surveillance systems report the use of CCs or discharge diagnoses based on ICD-9 codes. Most of these use discharge diagnosis ICD-9 codes in specialized settings such as the Military [ 13 ] or in HMOs [ 23 ]. ED ICD-9 codes were also used when processing data retrospectively [ 4 ]. In most EDs, however, ICD-9 discharge diagnosis coding is not performed on a "near-real" time basis and would not be available for "near-real time" surveillance. By contrast, free text chief complaints are obtained at the time of patient entry into all Emergency Departments and free text discharge diagnoses are determined at or close to the time of ED discharge. Therefore, use of our grouping scheme is relevant to the majority of EDs in which chief complaints and discharge diagnoses are recorded as free text and are available for "near-real time" surveillance. Because of their timeliness, CCs are used in our "near-real time" surveillance system B-SAFER [ 31 , 32 ]. We considered using CoCo, a naive Bayesian free-text classifier developed by the University of Pittsburgh [ 33 ], but this was not made available to us. Another automated classification based on weighted key words system is used by ESSENCE [ 34 ]. The New York City Department of Health uses a key word and key phrase SAS-based coding system [ 35 ]. A comparison of the performance of expert based classification systems such as ours, and automated classification systems has not been done. There are other potential limitations in using ICD-9 codes for surveillance. It is to be expected that early cases of unusual diseases will be misdiagnosed. Assigned ICD-9 diagnostic codes may be more reflective of the diagnostic bias or practice patterns of the provider, than of the true incidence of disease. Furthermore, ICD-9 diagnosis code assignment is potentially subject to billing bias: codes which garner the highest reimbursement may be used, rather then those that most accurately represent the disease process. Use of ICD-9 codes for chief complaints is also problematic. Because ICD-9 codes were developed for classification of diagnoses, the dictionary for chief complaints is not robust. Therefore, the use of free text chief complaints may result in increased sensitivity, although the B-SAFER team believes that some types of coding standards would be beneficial [ 36 ]. Results and applications Day-of-week patterns in EDs have been previously reported in the literature [ 4 , 26 , 27 , 37 , 38 ]. Although the magnitudes of the day-of-week effects vary depending on the setting, the first day of the work week typically exhibits the greatest number of events. And, as we have shown, there is day-of-week variability within infectious disease syndromes which can be obscured by the failure to consider each body system's pattern individually. It is also important to understand utilization patterns on weekends, which ED data provides, as the effects of bio-terrorist or natural outbreaks are unlikely to be limited to weekdays. Seasonal effects in infectious diseases are best known for respiratory infections (e.g. influenza and respiratory syncytial virus). This seasonality is in large part due to the yearly winter influenza epidemics. Our data is quite consistent with these findings. However, we provide an analysis of the pattern of respiratory related chief complaints based on the longest (8.6 years) historical data base. Furthermore, the seasonality of infectious disease complaints for other body systems, or for non-ID related complaints has not previously been reported. Seasonality in specific GI infections has been noted in other settings. Some gastrointestinal infections are more common in the winter (e.g. rotavirus) while others (e.g. Campylobacter , Cryptosporidium , enterovirus) are more common in the warm months from outdoor cooking and recreational water exposures. Seasonality is also important because the signal-to-noise ratio in complaint counts maychange depending on time of year. As illustrated in Figure 1 , the error bars are larger during the height of flu season than at other times of the year, leading to reduced sensitivity for detecting an increase in respiratory complaints at that time. It is unclear why, in our data, the number of respiratory complaints fell with time while the number of fever complaints rose. This may reflect the relative mildness of recent influenza seasons. Alternatively, rather than actual differences in patient presentations, this may represent changes in the practices of choosing or recording chief complaints. This would require further investigation. Implementation of a standard drop-down menu for chief complaints might prevent some bias over time in the selection of chief complaints. Use of a new system, however, would likely change the distribution of chief complaints and thus not allow for the creation of baselines based on historic data. Surveillance Using the models described above we successfully identified a respiratory outbreak in advance of the traditional flu-reporting data streams described in the Methods section. Incoming B-SAFER reports were monitored at least once daily, seven days a week, by the project epidemiologist. This allowed for prompt handling of events indicating a condition reportable by statute to the NM Department of Health. Because there is approximately a 2-week delay for traditional flu-related data sources, provided our respiratory CC captures some of the NM flu cases, we expected to, and did, identify a flu-related respiratory peak in advance of these other sources. Limitations This analysis is based on data from one ED and patterns identified may be somewhat specific to metropolitan Albuuerque. Indigent or Hispanic populations may be over-represented in the ED studied as compared to other EDs. Visit patterns may differ by the local health care infrastructure, population insurance status, access to care, or local climate. We wewere fortunate that electronic ED data was available for the previous eight years. Other institutions may lack the source data for a similar analysis. CCs are determined by a nurse and recorded in free text by a clerk. This process may conceivably distort patients' literal CCs. Free text CCs are quite variable and require extensive processing. These CCs may also have varied had they been recorded by a physician. Although the rationale for using CCs rather than discharge diagnoses was provided above, there is a tradeoff between the better timeliness of CC data and the better sensitivity of discharge diagnoses [ 39 ]. Note that our modelling is as easily applied to diagnoses codes as to chief complaints. Any approach to disease surveillance using either CCs or discharge diagnoses requires large numbers of symptomatic patients. Analyses based on such large-scale counts are unlikely to discover a small and geographically dispersed event such as the anthrax Anthrax outbreak of October 2001. As we work more with our data, we will understand it better. Opportunities exist for performing sensitivity analyses, comparing the baseline patterns for CCs to those for discharge diagnoses, and more thoroughly evaluating the performance of our signals as compared to existing standards. Conclusion We have demonstrated a robust statistical approach to characterize baseline data for ED visits. We demonstrated day-of-week, seasonal and long-term effects by infectious disease in grouped chief complaint categories. ED data provides information on daily visit patterns, rather than just 5-day-a-week patterns. Using respiratory complaints as an example, we have shown that these models when applied to "near real-time" surveillance data provide an early indicator of an anomaly. This increase in respiratory visits was identified early by a rise in Page's statistic. This anomaly corresponded to events detected later by more traditional methods. Understanding baseline patterns in ED data provides the ability to distinguish expected versus unexpected events during infectious disease surveillance. Competing interests The author(s) declare that they have no competing interests. Authors' contributions JB and EU provided medical advice from the ED and public health perspectives. They were responsible for choice and groupings of chief complaints. JB obtained IRB approvals and recruited sites. TB and RP are responsible for choice and implementation of statistical tests. DF provided computer systems, interface and architecture. EJ provided technical input, review and project direction. JB, EU, TB and RP collaboratively wrote the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555597.xml
545960	Clustering under the line graph transformation: application to reaction network	Background Many real networks can be understood as two complementary networks with two kind of nodes. This is the case of metabolic networks where the first network has chemical compounds as nodes and the second one has nodes as reactions. In general, the second network may be related to the first one by a technique called line graph transformation (i.e., edges in an initial network are transformed into nodes). Recently, the main topological properties of the metabolic networks have been properly described by means of a hierarchical model. While the chemical compound network has been classified as hierarchical network, a detailed study of the chemical reaction network had not been carried out. Results We have applied the line graph transformation to a hierarchical network and the degree-dependent clustering coefficient C ( k ) is calculated for the transformed network. C ( k ) indicates the probability that two nearest neighbours of a vertex of degree k are connected to each other. While C ( k ) follows the scaling law C ( k ) ~ k -1.1 for the initial hierarchical network, C ( k ) scales weakly as k 0.08 for the transformed network. This theoretical prediction was compared with the experimental data of chemical reactions from the KEGG database finding a good agreement. Conclusions The weak scaling found for the transformed network indicates that the reaction network can be identified as a degree-independent clustering network. By using this result, the hierarchical classification of the reaction network is discussed.	Background Recent studies on network science demonstrate that cellular networks are described by universal features, which are also present in non-biological complex systems, as for example social networks or WWW . Most networks encountered in real world have scale-free topology, in particular networks of fundamental elements of cells as proteins and chemical substrates [ 1 - 4 ]. In these networks, the distribution of node degree follows a power-law as P ( k ) ~ k - γ (i.e., frequency of the nodes that are connected to k other nodes). The degree of a node is the number of other nodes to which it is connected. One of the most successful models for explaining that scale-free topology was proposed by Barabási-Albert [ 5 ], which introduced a mean-field method to simulate the growth dynamics of individual nodes in a continuum theory framework. However, although that model was a milestone to understand the behavior of real complex networks, it could not reproduce all the observed features in real networks such as clustering dependence. The observed properties of networks with N nodes are: scale-free of degree distribution P ( k ) ~ k - γ , power-law scaling of clustering coefficient C ( k ) ~ k -1 and a high value for the average of the clustering coefficient < C > and its independence with network size. In particular, the dependence of C ( k ) ~ k -1 was one of the results obtained by [ 6 ]. In order to bring under a single framework all these observed properties in real networks Ravasz et al. (the RSMOB model in what follows) suggested successfully a hierarchical and modular topology [ 7 , 8 ]. In [ 8 ], a network with the above mentioned properties was called hierarchical network. We note that this deterministic model is an extension of the original model shown in [ 9 ]. It is also worth noticing that this modular topology was also suggested in biological networks by [ 10 , 11 ]. Interestingly, these properties of networks have been found in many non- biological and biological networks. One of them, which is the subject of our study, is the metabolic network. It is interesting to note that the metabolic network is an example of bipartite networks [ 12 ]. In a bipartite network there are two kinds of nodes and edges only connect nodes of different kinds. In the metabolic network these nodes are chemical compounds and reactions. The network generated by the chemical compounds (reactions) is called compound (reaction) projection. A line graph transformation (i.e., each edge between two nodes becomes a node of the transformed network) may relate both projections. However, although the line graph transformation works fine on bipartite networks, the transformed network (in the particular case of metabolic networks) may not be totally the same as the reaction projection. This issue is discussed in detail later. In addition, we will show by comparing with the experimental data, that this fact does not affect our qualitative results. Furthermore, a detailed analysis of the line graph transformation focused on the degree distribution P ( k ) and applied to some real networks can be found in [ 13 ]. In that work, similarities and differences between the line graph transformation and the metabolic network are also discussed. There it was found that if the initial network follows a power-law P ( k ) ~ k - γ , the transformed network preserves the scale-free topology and in most cases the exponent is increased by one unit as P ( k ) ~ k - γ +1 . It is also worth noting that the line graph transformation has recently been applied with success by Pereira-Leal et al. [ 15 ] on the protein interaction network with the aim to detect functional modules. In that work, the edges (interactions) between two proteins become the nodes of the transformed network (interaction network). By means of the line graph transformation, the interaction network has a higher clustering coefficient than the protein network. By using the TribeMCL algorithm [ 16 ] they are able to detect clusters in the more highly clustered interaction network. These clusters are transformed back to the initial protein-protein network to identify which proteins can form functional clusters. At this point, we note that the aim of our study is not to detect functional modules from the metabolic network. In our work the line graph transformation is used successfully to evoke general topological properties related to the clustering degree of the reaction network. The observed topological properties related to the clustering degree of the metabolic network (in particular, the chemical compound network) have been properly described by means of the RSMOB model. In the present work, our aim is to study the clustering coefficients C ( k ) and < C > of the reaction network by using two approaches: Firstly, we derive mathematical equations of those coefficients in the transformed network. Secondly, we apply the line graph transformation to a hierarchical network. The results from both methods are compared with experimental data of reactions from KEGG database [ 14 ] showing a good agreement. Though we started this work motivated by theoretical interest in the line graph transformation, the results provide explanation for the difference of C ( k ) between the compound network and the reaction network. In our work, the hierarchical network is generated by the RSMOB model, where the nodes correspond to chemical compounds and the edges correspond to reactions. While the RSMOB model reproduces successfully the hierarchical properties of the compound network, here we show that this hierarchical model also stores adequate information to reproduce the experimental data of the reaction network. Our study indicates that it is enough to apply the line graph transformation to the hierarchical network to extract that information. While C ( k ) follows the power-law k -1.1 for the initial hierarchical network (compound network), C ( k ) scales weakly as k 0.08 for the transformed network (reaction network). Consequently, we conclude that the reaction network may not be classified as a hierarchical network, as it is defined in [ 8 ]. Remark In [ 8 ], a network with scale-free topology, scaling law of C ( k ) ~ k -1 , and high degree of clustering was called hierarchical network . Consequently, the RSMOB model shown in [ 7 , 8 ] was developed to bring these properties under a single roof. Furthermore, in [ 7 , 8 , 17 ] some networks (in particular metabolic network) were classified as hierarchical network according to the above definition. To be precise, it was argued that the signature of the intrinsic hierarchy (or hierarhical modularity) is the scaling law of C ( k ). Moreover, in a more recent work [ 18 ], it was claimed that traditional random and scale-free models do not have a hierarchical topology because C ( k ) is independent of k (i.e., flat plot of C ( k )). In addition, analyses of C ( k ) were recently carried out in [ 19 ] to uncover the structural organization and hierarchy of non-biological weighted networks. At this point, we must note that we have followed the research done by Barabási et al , [ 7 , 8 , 18 ] and consequently, we have used its definition of hierarchical network in the present work. However, it is also worth noticing that another way to quantify the hierarchical topology of a network is recently introduced by [ 20 , 21 ]. It is based on the concept of a hierarchical path : a path between nodes i and j is called hierarchical if (1) the node degrees grow monotonously (" up path "), and it is followed by a path where the node degrees decrease monotonously (" down path ") or (2) the node degrees along this path changes monotonously from one node to the other. The fraction of shortest paths in a network, which are also hierarchical paths is called H . If H is very close to 1, the network shows a hierarchical organization. This definition seems interesting, and consequently, as a future work it would be worth to examine some biological networks (in particular, metabolic networks) by using this approach. One remark about this concept is that it focuses on hierarchy and may not contain enough information about modularity or clustering. For a brief discussion of these issues, we refer to some very useful notes written by Dorogovtsev et al. [ 21 ] (see also [ 22 ] for further information about related topics in networks). Results and discussion Clustering coefficients C ( k ) and < C > Recent analyses have demonstrated that the metabolic network has a hierarchical organization, with properties as: scale-free degree distribution P ( k ) ~ k - γ , power-law dependence of clustering coefficient C ( k ) ~ k -1 and independence with network size of the average clustering coefficient < C >, where N is the total number of nodes in a network [ 7 ]. The clustering coefficient can be defined for each node i as: where n i denotes the number of edges connecting the k i nearest neighbors of node i to each other, C i is equal to 1 for a node at the center of a fully interlinked cluster, and it is 0 for a node that is a part of a loosely connected cluster [ 7 ]. An example can be seen in Fig. 1A . Figure 1 (A) Example of clustering in an undirected network. Continuous and dash-dotted lines mean interaction between nodes. In addition, the dash-dotted line defines the only triangle where the node 1 (red) is one of the vertices. The node 1 has 4 neighbors ( k i = 4), and among these neighbors only one pair is connected ( n 1 = 1). The total number of possible triangles that could go through node i is 6. Thus, the clustering coefficient has the value C 1 = 1/6. High density of triangles means high clustering coefficient. (B) We show an example of the line graph transformation. The initial graph G corresponds to one subgraph which belongs to the Lysine Biosynthesis metabolic pathway. This graph is constructed by taking nodes as chemical compounds and edges as reactions. By applying the line graph transformation we find graph L ( G ), which is the reaction graph embedded in the graph G . The nodes of the graph L(G) are the reactions of the graph G [13]. Geometrically, n i gives the number of triangles that go through node i . The factor k i ( k i - 1)/2 gives the total number of triangles that could go through node i (i.e., total number of triangles obtained when all the neighbors of node i are connected to each other). In the case of Fig. 1A , there is one triangle that contains node 1 (dash-dotted lines), and a total of 6 triangles could be generated as the maximum. Hence, the clustering coefficient of node 1 is C 1 = 1/6. On the other hand, the average clustering coefficient < C > characterizes the overall tendency of nodes to form clusters as a function of the total size of the network N . The mathematical expression is: The structure of the network is given by the function C ( k ), which is defined as the average clustering coefficient over nodes with the same node degree k . This function is written as: where N k is the number of nodes with degree k , and the sum runs over the N k nodes with degree k . A scaling law k -1 for this magnitude is an indication of the hierarchical topology of a network. Once the theoretical definitions have been introduced, our aim is to analyse how the coefficients < C > and C ( k ) are modified under the line graph transformation. Line graph transformation to metabolic networks: spurious nodes Given an undirected graph G , defined by a set of nodes V ( G ) and a set of edges E ( G ), we associate another graph L ( G ), called the line graph of G , in which V ( L ( G )) = E ( G ), and two nodes are adjacent if and only if they have a common endpoint in G (i.e., E ( L ( G )) = {{( u , v ), ( v , w )}\|( u , v ) ∈ E ( G ), ( v , w ) ∈ E ( G )}). This construction of graph L ( G ) from the initial graph G is called line graph transformation [ 23 ]. It is worth noting that in a previous work [ 13 ] the degree distribution P ( k ) was studied by applying line graph transformation to synthetic and real networks. There it is assumed an initial graph G with scale-free topology as P ( k ) ≈ k - γ . As the degree of each transformed node (i.e., an edge in G ) will be roughly around k , the distribution of the line graph L ( G ) should be k · k - γ = k - γ +1 with degree around k . Therefore, it is concluded that if we have a graph G with a probability distribution following a power-law as k - γ , then L ( G ) will follow a power-law as k - γ +1 . The real networks under study were protein-protein interaction, WWW , and metabolic networks. In Fig. 1B , we can see an example of the line graph transformation applied to a subgraph of the metabolic network. However, it is important to point out one issue. In metabolic networks, there are cases where spurious nodes appear (see Fig. 2 ). For example, we consider two reactions sharing the same substrate (or product) and at least one of the chemical reaction has more than one product (or substrate). If we apply a line graph transformation to this network, we would obtain more than two nodes in the transformed network, where only two nodes (reflecting two reactions present in the real process) should appear. These spurious nodes appear only when one (or some) reaction(s) in the network has more than one product (or substrate). Therefore, these cases should be computed and transformed by generating only as many nodes in the transformed network as reactions in the real metabolic process. This procedure is called physical line graph transformation. In the present work, we have applied this procedure to generate the reaction network by using experimental data from the KEGG database. Experimental data are shown later in Figs. 7B and 8 (blue diamonds). More detailed information about this issue can be found in [ 13 ]. Figure 2 We show two reactions ( R1 , R2 ) sharing a common chemical compound, and both reactions contain more than one product (or substrate). The substrate graph G (chemical compounds) is shown dark blue circles. The reaction graph L real (reactions) is shown with light red circles. If we apply a line graph transformation to this network, we would obtain more than two nodes in the transformed network. However, only two nodes (reactions) are present in the real process. These cases are computed and transformed by generating only as many nodes in the transformed network as reactions in the real metabolic process. We call this procedure physical line graph transformation. Equations of C ( k ) and < C > under the line graph transformation We assume a graph G as it is depicted in Fig. 3A . In this graph, edge a connects two nodes with degree k ' and k ". We apply the line graph transformation to this graph G and the result of this transformation is the line graph of G , L ( G ) shown in Fig. 3B . We see that, under the line graph transformation, the nodes of L ( G ) are the edges of G , with two nodes of L ( G ) adjacent whenever the corresponding edges of G are. Figure 3 (A) Graph G with two hubs with degree k ' and k " connected by edge a . (B) The corresponding line graph L ( G ) after the line graph transformation is done. (C) Graph G where edges b and b ' have a common node as endpoint. (D) Line graph of (C). It is worth noticing that (D) has only one more edge than (B). Hence, (D) has one more triangle that go through node a than (B). The clustering coefficient for the node a in the transformed network can be written by using Eq. (1) as: where k = k ' + k " - 2, because the edge a vanishes in the graph L ( G ). This equation ignores cases where edges in the graph G , b and b ' for example, have a common node as endpoint (i.e., existence of triangles or loops in Fig. 3C ). However, we can quantify these cases by using a new parameter l . As we can see in Fig. 3C–D , edges with one common node as endpoint in the graph G means one additional edge in the graph L ( G ). This additional edge in L ( G ) connects two neighbors of node a . By following definition of Eq. (1), it means that n a increases its value by one unit. We can consider these cases by increasing one unit the parameter l for each common node as endpoint of two edges in the graph G (for example, l = 1 means one common node). We write Eq. (4) after introducing the parameter l as: where if l = 0 means that there are not loops and we recover Eq. (4). Though it is more realistic to consider the parameter l as a function of k ' and k ", we have considered l as an independent parameter. However, this simplification does not affect the qualitative features of our results. It should be noted that l always contributes to increasing the value of C a ( k ) and C a ( k ) ≤ 1 always holds from the definition. In order to study the limits of Eq. (5) we consider the following two cases: • a ) k ' = k ": We analyse the case where both degrees have the same value. We also consider the cases when l = 0 and l ≠ 0 in order to study the effect of triangles. We show the results in Fig. 4 . For large k ', Eq. (5) goes asymptotically to 1/2 for l = 0 and l ≠ 0. We also see that for k ' ≥ 25, all lines are very close to 1/2. For low k ' and l = 0, C a ( k ) takes values from 0.33 ( k ' = 3) to 0.48 ( k ' = 20). Hence, we see in Fig. 4 that higher values of l (more triangles) increase the values of C a ( k ). Figure 4 Values of C a ( k ) from Eq. (5) calculated by taking k ' = k ". Number of common nodes as endpoint of two edges (triangles) are indicated by the parameter l . The degree of transformed nodes is k = k ' + k " - 2 because the edge a vanishes in the graph L ( G ). • b ) k " = constant, k ' >> k ": We plot in Fig. 5 three cases. k " is fixed with constant values as k " = 5 (black), k " = 10 (red), k " = 20 (blue) and k ' is a free parameter. We see that C a ( k ) approaches to 1 when k ' takes large values. For low k ', the case k " = 5 shows a minimum with a few values of k ' below 1/2. As we can see with dotted and dash-dotted lines in Fig. 5 , the presence of triangles ( l ≠ 0) increases the value of C a ( k ). Finally, for k " = 10 and k " = 20, we see that only a few values of C a ( k ) are slightly below 1/2 for low k '. This analysis is complemented by calculating the minimum value of C a ( k ) analytically as: . The value of k ', where the function C a ( k ) takes the minimum value, is given by: Figure 5 Values of C a ( k ) from Eq. (5) calculated by taking k " with constant values as k " = 5 (black line), k " = 10 (red), k " = 20 (blue) and k ' as a free parameter. Dotted and dash-dotted lines show the presence of triangles ( l ≠ 0). Triangles increase the value of C a ( k ). where positive solution of the square root is written. By substituting this equation into Eq. (5), it is possible to calculate the minimum value of C a ( k ) for each configuration of l and k ". From these two cases, we can conclude that for hubs (i.e., those nodes with high degree ( k ' and k " >> 1)) and for highly clustered networks (many triangles l >> 1), the values of C a ( k ) in the transformed network are between around [ , 1]. To calculate the distribution of C ( k ) in the transformed space ( C T ( k )) we introduce the concept of assortativity. By assortative (disassortative) mixing in networks we understand the preference for nodes with high degree to connect to other high (low) degree nodes [ 24 ]. By following Newman [ 24 ], we define the probability distribution to choose a randomly edge with two nodes at either end with degrees k ' and k " as e k ' k" . We also assume that the nodes of the initial network are following a power-law distribution k - γ and have no assortative mixing. Under these assumptions, the probability distribution e k ' k" of edges that link together nodes with degree k ' + k " can be written as: We make a convolution between Eq. (4) and Eq. (7), by summing for all the possible degrees of the two nodes at either end of edges ( k ', k "), which can generate transformed nodes with degree k = k ' + k " - 2. Thus, we obtain: According to the structure of C T ( k ) and the behavior of C a ( k ) exposed above, C T ( k ) will grow smoothly for large k , i.e., scaling weakly with the node degree k . We have calculated numerically this expression and the results are discussed later in Fig. 7 . We have also calculated the analytical expression for < C >, and we have found that < C > has a size-independent behavior before and after the line graph transformation is done. We can write the number of nodes with degree k as: and we assume that C ( k ) = A · k - α , where A is a constant. This constant changes when we consider hierarchical networks with different number of nodes in the initial cluster [ 7 ]. But it seems natural because in that case the degree distribution P ( k ) ~ k - γ of the network also changes. For < C > before the transformation we can write: Note that the summation in the denominator begins with k = 1 because we renormalize over all the probability distribution. Furthermore, we can obtain < C > by using the RSMOB model (explained in next section in detail). This model starts by generating a fully connected cluster of m nodes, such that the connectivity of each node is k = m - 1. In the following iteration, m - 1 replicas of the initial cluster are generated, and linked to the central node of the original cluster in such a way that the central node of the original cluster gains ( m - 1)·( m - 1) edges, and its total connectivity being k = m - 1 + ( m - 1) 2 . By iterating these procedure, it is easy to see that hubs (i.e., central nodes of each replica) will have connectivities , with j = 1, ..., log m N being the iteration number. Therefore, assuming that the degree distribution P ( k ) and the clustering coefficient C ( k ) are power-laws with exponents γ ' and α respectively, the expression for < C > for the hubs reads as: where A ' is a constant adjusted so that < C >=1 holds for j = 1. The upper limit of the summation log m N is obtained by means of the expression m j = N , which gives the total number of nodes in the network and denotes the exponent of the power-law distribution of hubs in the RSMOB model. We must note that in a hierarchical network, the number of nodes with different degree k is scarce, therefore the probability distribution of node degree is properly defined as P ( k ) = (1/ N tot )( N k /Δ k ), where N k is the number of nodes with degree k , N tot is the total number of nodes, and Δ k means that nodes with degree k are binned into intervals. In addition, we note that for the hierarchical model, Δ k changes linearly with k . Hence, the exponent of the power-law is given by γ = 1 + γ ', with where m is the number of nodes in the initial module. By using Eqs. (10) and (11), we will see later (Tables 1 and 2 ) that < C > converges to a constant. In order to calculate < C > after the line graph transformation is applied (< C T >), we make the substitution C ( k ) → C T ( k ) in Eq. (10). As from Eq. (8) we have seen that C T ( k ) is almost constant, we can conclude that < C T > also has a constant behavior and it is almost independent with network size. While the scaling law of C ( k ) ~ k -1 was proved mathematically in [ 6 ], here we have obtained the analytical expressions of C T ( k ), < C > and < C T >. Table 1 Results of < C > evaluated by using Eq. (10) and the needed parameters in that calculation for 3 different setups: γ = 1 + γ ', where ( P ( k ) ~ k - γ ), α ( C ( k ) ~ k - α ), A ( C ( k ) = A · k - α ). Eq. (10) is a general expression of < C >. m initial nodes γ α A < C > 3 2.58 1.1 2.34 0.20 4 2.26 1.1 3.68 0.36 5 2.16 1.1 5.18 0.54 Table 2 Results of < C > evaluated by using Eq. (11) for 3 different setups. The exponent of the power-law distribution of hubs is given by . The parameter α has same meaning as in Table 1. We also notice that in Eq. (11), A ' is adjusted so that < C >=1 holds for j = 1. Eq. (11) is the particular expression of < C > applied to the RSMOB model. m initial nodes γ ' α < C > (Eq. (11) 3 1.58 1.1 0.78 4 1.26 1.1 0.81 5 1.16 1.1 0.83 Line graph transformation to a hierarchical network: numerical results The RSMOB model [ 8 ] is able to reproduce the main topological features of the metabolic network. We follow the method described in [ 8 ] and generate a hierarchical network. Then, we apply the line graph transformation to that network. Fig. 6 illustrates the hierarchical network generated by the RSMOB model. The network is made of densely linked 5-node modules (it is worth noticing that the number of nodes in the initial module can be different than 5) that are assembled into larger 25-node modules (iteration n = 1, 5 2 = 25 nodes). In the next step four replicas are created and the peripheral nodes are connected again to produce 125-node modules (iteration n = 2, 5 3 = 125 nodes). This process can be repeated indefinitely [ 8 ]. To evaluate C ( k ), we have constructed three hierarchical networks with 3, 4, and 5 initial number of nodes. These networks were generated up to 7 (6561), 5 (4096), and 4 (3125) iterations (nodes), repectively. Once we have constructed these three networks, we apply the line graph transformation to them, and we calculate the C T ( k ) clustering coefficient for the transformed networks. In Fig. 7A we show the results of the clustering coefficient of the transformed network. Circles, triangles and squares indicate the values of C T ( k ) for the transformed network with 3, 4, and 5 initial nodes, respectively. In Fig. 7A we also plot with continuous lines the values of C T ( k ) obtained from Eq. (8). From top to bottom the lines correspond to the networks of 3, 4 and 5 initial nodes, respectively. In Fig. 7A , we see that the lines show an acceptable agreement with the overall tendency of data generated by the transformed network. In Fig. 7B , we see that the results from theoretical calculation of C T ( k ) via Eq. (8) (lines) are in good agreement with the experimental data (diamonds) from the KEGG database [ 14 ]. Moreover, in order to have enough statistics to compare with the analytical expression for the C T ( k ), we have binned into seven intervals the experimental data according to degree k (1 < k ≤ 8 < ,..., 128 < k ≤ 256, 256 < k ≤ 512), and averaged over the C T ( k )'s obtained in that range (red circles). It shows a better agreement between KEGG results and the analytical curves. The only disagreement comes at k = 2. This is easy to understand because in the hierarchical model depicted in Fig. 6 , we can only find C ( k = 2) = 1 for 3 initial nodes by construction of the network. However, in real networks, we could find nodes which have only two neighbors and, in some cases, these neighbors could be connected. In these cases the clustering coefficient takes value one. In Fig. 8 , we show the results for C T ( k ) after the line graph transformation is applied to the hierarchical network generated by 4 initial nodes and up to 5 iterations. The results are shown with empty triangles (red) and fitted to the dashed line. We see that C ( k ) ~ k -1.1 changed into C T ( k ) ~ k 0.08 . We also see that the line graph transformation increases the average of the clustering value of the transformed network. These theoretical results were compared with the experimental data from KEGG [ 14 ], finding a good agreement, and supporting the result of a degree-independent clustering coefficient C T ( k ) for the reaction network. Figure 6 Hierarchical network generated by using the RSMOB model [8]. Starting from a fully connected cluster of 5 nodes, 4 identical replicas are created, obtaining a network of N = 25 nodes in the first iteration n = 1 (5 2 = 25 nodes). We have linked to each other the central hubs of the replicas by following [7]. This process can repeated indefinitely. We note that the initial number of nodes can be different than 5. Figure 7 (A) We plot the results of the hierarchical model for C T ( k ) for different configurations. 3 initial nodes and up to 7 iterations (circles), 4 initial nodes and up to 5 iterations (triangles), 5 initial nodes and up to 4 iterations (squares). Prom top to bottom (3 initial nodes (black), 4 initial nodes (red), 5 initial nodes (green)), we show with lines the results of C T ( k ) obtained by means of Eq. (8). (B) The lines have the same meaning as before and the diamonds correspond to the experimental data for reactions from the KEGG database [14]. Experimental data involves 163 organisms. Circles (red): Experimental data binned into seven intervals according to degree (1 < k ≤ 8 <, ..., 128 < k ≤ 256, 256 < k ≤ 512). Figures in log-linear scale. Figure 8 Full circles (red) and dot-dashed line (red): C ( k ) evaluated with the hierarchical network. Empty triangles (red) and dashed line (red): C ( k ) after the line graph transformation is done over the hierarchical network ( C T ( k )). Diamonds (blue): C T ( k ) of reactions data from the KEGG database [14]. Empty circles (blue) and continuous line: C ( k ) of compounds data from KEGG. Hierarchical model with 4 initial nodes and 5 iterations. Figure in log-log scale. For < C > we have evaluated Eq. (10) for 3 different configurations. We have considered 3 initial nodes, 4 and 5 initial nodes nodes up to 7, 5 and 4 iterations, respectively. As it is explained in [ 7 ], < C > approaches asymptotically to a constant value, being independent of the size of the network. The asymptotic value depends on the initial number of nodes. We calculated the values of γ corresponding to the degree distribution P ( k ) ~ k - γ for each network, and the related constant A , which appears in Eq. (10). We show in Table 1 the values of these parameters and the results of < C > obtained by Eq. (10). These values, as it can be seen in Fig. 9A , are below the asymptotic values of ~ 0.66 (circles) and ~ 0.74 (triangles) obtained by using the RSMOB model. However, we have found an explanation for this result. In Fig. 8 , the full circles at the top of the dash-dotted line correspond to non-hubs nodes. We have checked that these nodes do not follow a power-law, hence the value of C ( k ) is being overestimated by the scaling dependence k -1 and it provides a larger value of < C >. In [ 7 ], the values of < C > from hierarchical model were compared with the experimental values of 43 organisms. The values of < C > for each organism were around 0.15 – 0.25. By using the KEGG database we have evaluated the experimental value < C > for 163 organisms and we obtained an average value of 0.08. Figure 9 Dark (black): < C > is calculated by using the hierarchical network. Light (green): < C T > (< C > after the line graph transformation is applied to the hierarchical network). Circles (3 initial nodes), Triangles (4 initial nodes). Star (red): Experimental < C T > for reactions from the KEGG database [14]. (B) < C > is calculated by using Eq. (11). The results show a good agreement and similar tendency to those shown in Fig. 9(a) (dark circles and triangles). Figures in log-linear scale. We show in Fig. 9A the values of < C > calculated for networks generated by 3 initial nodes (circles) and 4 initial nodes (triangles) by using the RSMOB model. We see that < C > approaches asymptotically to constant values around ~ 0.66 (circles) and ~ 0.74 (triangles), being independent of the size of the network. Once the line graph transformation is applied, we see that the corresponding values of < C T > also approach asymptotically to constant values. Hence, < C T > also is size-independent for large N (empty circles and triangles). In addition, we have averaged the experimental value of the clustering coefficient for reactions of 163 organisms found in KEGG database and we have obtained the value of < C T >= 0.74. We see that the experimental value < C T > for reactions is in good agreement with the asymptotic values obtained by the transformed network (empty triangles and circles). Furthermore, we have also calculated < C > by using Eq. (11). This equation should reproduce the results of < C > calculated by using the RSMOB model (dark circles and triangles in Fig. 9A ). In Fig. 9B , we see that the results are qualitatively similar to those shown in Fig. 9A (dark circles and triangles). We remark that the theoretical analysis of < C > and < C T > done here has also been useful to prove that they are independent of network size. Finally, in Fig. 10 we plot the hierarchical network (left) and the transformed network (right) by using the graph drawing tool Pajek [ 25 ]. We see the high degree of compactness of the transformed network. It could be related to the concept of robustness of a network. It means that by removing one node randomly from the reaction network depicted in the Fig. 10 , the normal behavior of the cell might be preserved by finding an alternative path (reaction) to complete the task. This fact could be a consequence of the high degree of clustering and connectivity between the nodes in the transformed network. Figure 10 (A) Hierarchical network generated by using the model of ref. [8] with 4-node modules and up to 2 iterations. (B) Network after the line graph transformation. We see a huge interlinked cluster in the center of figure, which generates the degree-independent clustering coefficient C T ( k ) (it scales weakly as C T ( k ) ~ k 0.08 ). Conclusions We have studied here the clustering coefficients C ( k ) and < C > of the reaction network by applying the line graph transformation to a hierarchical network. This hierarchical network was generated by using the RSMOB model, which reproduces properly the topological features of the metabolic network, in particular the compound network. Our results indicate that by applying the line graph transformation to the hierarchical network, it is possible to extract topological properties of the reaction network, which is embedded in the metabolic network. The RSMOB model stores the adequate information of the reaction network and the line graph transformation is one useful technique to evoke it. While C ( k ) scales as k -1.1 for the initial hierarchical network (compound network), we find C ( k ) ~ k 0.08 for the transformed network (reaction network). This theoretical prediction was compared with the experimental data from the KEGG database, finding a good agreement. Our results indicate that the reaction network is a degree-independent clustering network. Furthermore, the weak scaling of C ( k ) for the reaction network suggests us that this network may not have hierarchical organization. However, further analyses of this network, and in general of all biological networks, by following the concept of hierarchical path are encouraged [ 20 , 21 ]. On the other hand, we have also conducted an analytical derivation for the clustering coefficients C ( k ) and < C >. Expressions for these coefficients were calculated before and after the line graph transformation is applied to the hierarchical network. The agreement obtained by using these expressions was found acceptable, and consequently, they could be useful for further analyses in different networks (biological and non-biological). The line graph transformation has recently been applied on metabolic networks [ 13 ] to study the scale-free topology of the reaction network, and on the protein-protein interaction network to detect functional clusters [ 15 ]. The work done here is another important application of this interesting technique. Authors' contributions JCN conceived of the study, designed and implemented the analyses, and prepared the manuscript. NU carried out computational implementations and experiments. TY participated in the acquisition and processing of data from KEGG database. MK provided conceptual guidance and data from the KEGG database, and conceived the initial idea of the two complementary metabolic networks. TA provided guidance, coordinated and participated in the biological and theoretical analyses, and revised the manuscript. All authors read and approved the final manuscript. Table 3 Definitions of functions and their values before and after the line graph transformation is applied to the hierarchical network. N k : number of nodes of degree k . The † symbol means that these dependences were analyzed in the present work, while the * symbol means that it was studied in our previous work [13]. Func. Definition Dependence before Dependence after (Eq. (11) P ( k ) N k / N k - γ k - γ +1 * C i ( k ) 2 n /[ k i ( k i - 1)] k -1.1 k 0.08† < C > size-independent † size-independent †	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545960.xml
555568	Necrotic gangrenous intrathoracic appendix in a marfanoid adult patient: a case report	Background A diaphragmatic hernia is defined as a defect in part of the diaphragm through which abdominal contents can protrude into the thorax. It may be congenital or acquired. In this case report, we aim to demonstrate a congenital diaphragmatic hernia in an adult marfanoid patient which required emergency treatment Case presentation A 43 year old woman was admitted with classical appendicitis requiring surgery. She incidentally had Marfan's clinical features with a positive family history for the syndrome. At operation she had grossly abnormal abdominal anatomy. Radiological investigations demonstrated a large right congenital diaphragmatic hernia with an intrathoracic hernial sac containing a perforated gangrenous appendix. The hernial sac was opened surgically and the appendix excised. The patient made a full recovery. Conclusion Diaphragmatic hernias are usually congenital in nature often requiring early corrective surgery for future survival. We have demonstrated the presence of an unusually large diaphragmatic defect, almost a hemidiaphragmatic defect, of unknown direct etiology, but of some possible association with Marfan's syndrome in an adult patient presenting with an acute perforated gangrenous appendix requiring emergency life-saving surgery.	Background A diaphragmatic hernia is defined as a defect in part of the diaphragm through which abdominal contents can protrude into the thorax. It may be congenital or acquired, usually through trauma [ 1 ]. Congenital diaphragmatic hernias usually occur in the posterolateral portion of the diaphragm (Bochdalek's hernia) and are on the left side in 90% of cases. Loops of bowel, even most of the abdominal contents, may protrude into the hemithorax on the involved side [ 1 , 5 ]. Most congenital diaphragmatic hernias are detected in the newborn whereby, after delivery, as the infant cries and swallows air, the loops of bowel quickly fill with air and rapidly enlarge, causing acute respiratory embarrassment as the heart and mediastinal structures are pushed to the right, compressing the right lung. Respiratory distress is immediate in severe cases; a scaphoid abdomen (due to displacement of abdominal viscera into the chest) is likely. Bowel sounds (and an absence of breath sounds) may be heard over the involved hemithorax. In less severe cases, mild respiratory difficulty develops a few hours or days later as abdominal contents progressively herniate through a smaller diaphragmatic defect [ 1 ]. Urgent surgery is usually required to repair the defect [ 1 ]. Acquired diaphragmatic hernias are relatively rare and result from either blunt or penetrating trauma. Blunt trauma typically produces large radial tears measuring 5–15 cm, most often at the posterolateral aspect of the diaphragm. Penetrating injuries to the diaphragm can follow accidental trauma, knife or gunshot wounds. Typically, the defect is small, less than two centimeters in size and may present late after years of gradual herniation and enlargement. Occasionally a shotgun blast causes a large defect [ 3 , 4 ]. Diaphragmatic hernias are usually congenital in nature often requiring early corrective surgery for future survival. There have been three recent publications [ 5 - 7 ] in the literature relating Marfan's syndrome to right sided diaphragmatic hernias. Jacobs et al [ 5 ] described an association between unique FBN1 gene mutations in neonates and the presence of large unilateral diaphragmatic hernias, while Subirats et al [ 7 ] demonstrated a correlation between patients exhibiting positive Marfan's syndrome features and having unilateral diaphragmatic hernias. Yetman et al [ 5 ] recently described a case of acute dyspnoea in a child with Marfan's syndrome secondary to bowel herniation into the thoracic cavity. In these published cases, the diaphragmatic hernias were described in neonates or children and always required corrective surgery for survival, unsuccessfully in two cases. Case presentation A 43 year old female district nurse presented to the accident and emergency department with a 6 hour history of initially generalized abdominal pain which was localizing to the right. This was associated with nausea and loss of appetite, made worse by movement and not relieved with intramuscular morphine injection. There was no history of recent or past trauma to the chest or abdomen. On examination, she was tender in the right upper quadrant and right iliac fossa with rebound and guarding, with a Rovsing positive sign and normal bowel sounds. She was incidentally found to have some marfanoid features including the long span of upper limbs, a high arched palate and the very soft early diastolic murmur of aortic regurgitation. The patient had a positive family history of Marfan's syndrome but had never undergone genetic testing to confirm the diagnosis. A likely diagnosis of appendicitis was made on the clinical picture associated with a pyrexia and raised white cell count and C – reactive protein. Her abdominal X-ray at that time showed absence of gas in the right side of her bowel. Her chest X-ray did not show any obvious abnormality (Figure 1 ) Figure 1 Chest radiograph demonstrating no obvious abnormality She was taken to theatre the same day for appendicectomy through a standard right iliac fossa incision. There was a small amount of free fluid but the right iliac fossa was filled with a normal looking gall bladder and small bowel only with no sign of the caecum, appendix, ascending or proximal transverse colon. The terminal ileum was found to run up under the right lobe of the liver towards the hilum. The right iliac fossa wound was closed and we proceeded to an exploratory laparoscopy. At laparoscopy, the liver was retracted to see under the right liver lobe. The proximal transverse colon was found to be running up towards the liver hilum were it felt to be tethered. This suggested a possible herniation of the right colon into the chest. The procedure was abandoned and a CT thorax and abdomen were performed to define the anatomy involved. This (Figure 2 , 3 ) demonstrated bilateral pleural effusions of moderate size with underlying unexpanded lungs. The liver had what appeared to be a large Reidl's lobe. Behind the right liver lobe, the right kidney was markedly elevated and the ileo-caecal junction appeared to lie between the liver and the kidney. The right side of the colon appeared to lie above the liver. Three dimensional reconstruction of the scans demonstrated absence of the right hemidiaphragm. (Figure 4 , 5 ) Figure 2 CT scan of thorax and abdomen demonstrating the abnormal anatomy Figure 3 CT scan of thorax and abdomen demonstrating the abnormal anatomy Figure 4 Three-dimensional CT reconstruction demonstrating the diaphragmatic defect Figure 5 Three-dimensional CT reconstruction demonstrating the diaphragmatic defect The symptoms and signs persisted and a laparotomy was performed. At laparotomy, there was some free fluid. There was definitely a large defect in the right hemidiaphragm. The right liver lobe had grown right up into the right intrathoracic space. There was a large hernial sac beneath the right lobe of the liver between the common bile duct, duodenum and liver, displacing the right kidney medially and containing transverse colon, terminal ileum, caecum, appendix and free fluid. The contents of this sac were successfully reduced and a perforated gangrenous appendix with pus was found within the intrathoracic hernial sac. A standard appendicectomy was performed. The right colon was formally mobilized, fully reduced and the caecum was fixed within the right iliac fossa. The patient was admitted to the intensive care unit postoperatively where she made a good recovery. She had instant relief of her abdominal symptoms and post-operative contrast study demonstrated the presence of the right colon within the abdomen (Figure 6 ). She was discharged a week post-operatively. Figure 6 Abdominal radiograph demonstrating the large colon present in the abdominal cavity Conclusion We have demonstrated a new case of an adult patient, with Marfan's syndrome clinical features and a positive family history of the syndrome, presenting with a large congenital diaphragmatic hernia, compatible with life and undiagnosed into adulthood, requiring an emergency admission into hospital with a perforated gangrenous intrathoracic appendix. Abbreviations CT = computerized tomography Competing interests The author(s) declare that they have no competing interests. Authors' contributions MJB designed the case report, researched the article and drafted the manuscript. Both MJB and JHV carried out the surgery and were involved in all investigations. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555568.xml
545784	How biologically relevant are interaction-based modules in protein networks?	The authors present a method to identify modules within protein-interaction networks. Phylogenetic profiles are used to determine the biological relevance of the modules.	Background There is a strong belief underpinning systems biology that between the individual molecules and an organism's phenotype there exist intermediary levels of organization [ 1 ]. The lowest level, and one that can be objectively defined, is that of the motif, for example a feedforward loop [ 2 - 5 ]. At the next level there exist putative modules within networks [ 6 - 16 ]. However, unlike motifs, modules are not objectively defined and are hence rather fuzzy. Moreover, even if a stringent definition or sophisticated algorithms could be envisaged, the data used to identify such modules are typically very noisy, for example, protein-protein interaction data. The central problem [ 17 ] with the notion of modules, therefore, is not identifying putative candidates but verifying which of them really reflect an important level of biological organization, rather than artifacts of the data or module-defining protocol. In addition, it would be of interest to determine the minimal information needed to identify such candidates, so that this level of organization can be readily probed, even in relatively poorly characterized systems. Given that we could define such modules for a particular data source, for example, protein-protein interactions, there exists the further problem of understanding how modules relate to other forms of organization. Do for example, the proteins in a given module within a protein-protein interaction network show evidence of being coexpressed? Are they regulated by the same transcription factors and do they have the same level of dispensability? Whether we can define modules in a stringent biologically relevant fashion is not just important for our understanding of the organization of biological systems. Many authors have conjectured that if modules are real they may also be more likely to contain proteins that are essential to viability. Hence, a network approach could be imagined to hone down potential drug targets such as, for instance, candidate targets for antimicrobials. Here we ask whether phylogenetic information could be used to verify putative interaction-based modules. The assumption we make is that if a set of proteins belongs to the same module and that module has some biological relevance, then such a set should be generally conserved to act as an integrated functional unit [ 18 , 19 ]. Hence we should expect a genome to contain roughly all the set components or none. The extent to which we find the module components present or absent together we define as the 'phylogenetic correlation' of the module. We show that this correlation can be used to verify putative modules in a network context and that the modules identified in this way have important biological properties. Results and discussion Extracting modules in protein networks Several network-clustering algorithms have been developed recently that make use of the local and global properties of networks [ 9 - 11 ]. To this end, it is helpful to represent networks as graphs, with proteins playing the role of nodes and protein-protein interactions playing the role of edges between nodes. In such graphs, the presence of modular topology could be manifested in the fact that the shortest distance, L , between any given node and the rest of the nodes in the graph would exhibit a similar pattern for those nodes belonging to the same module. Alternatively, modularity could also imply that proteins within a module would interact more frequently with each other than with proteins of different modules, a property characterized by high values of a generalized clustering coefficient, C (see Materials and methods). We introduce here a simple algorithm that makes use of both sources of information. The basic steps of the so-called overlap algorithm are as follows (see also Materials and methods and Figure 1a ). Selection of the number of modules C -based and L -based matrices were obtained from the interaction matrix. These matrices are the input data of a standard hierarchical agglomerative average-linkage clustering algorithm with a Pearson-based distance metric [ 20 ]. We obtained as an output of the clustering different sets of modules associated to each matrix by delimiting clusters according to a given number of branches present in the clustering tree ( ) (discarding those ones containing just a single protein). In the next step we calculated an average overlap between both modular structures. A -value with significantly high maximal overlap was then chosen. Extraction of a particular modular structure Having obtained C -based and L -based modules with a -value selected as previously described, we calculated the overlap of each C -based module with all those obtained with the L -based method. An L -based method less efficiently discriminates modular structures in small-world networks [ 21 ], collapsing some of the modules extracted with the C -based technique into a unique module. The C -based method is more robust but is weak at discriminating modules when organization levels are high. Therefore we used the C -based results as a template and the L -based method as a filter in the extraction of modular structure. In the C -based modular structure we kept in each module only those components which also appeared in the corresponding L -based module with which the selected C -module had the greatest overlap. In those cases with more than one module with maximal overlap, we selected one of them at random. Although finding the optimal classification choice is a common problem of clustering analysis, this simple algorithm allows one to select a -value with a high average maximal overlap and low overlap ratios between both methods, a measure of the reliability of the obtained modules (see Materials and methods and Additional data file 1 for more details). The overlap method was applied to the yeast protein-interaction network; that is, yeast would act as an imaginary 'poorly' characterized system where we can, however, check the relevance of our findings. This was derived from two public databases (see Materials and methods) and would be, more generally, the result of high-throughput experiments. In any case, these data are probably incomplete and no doubt contain false interactions [ 22 ]. Should the analysis be done on the whole network? Certainly this could be done - and many similar analyses have been done. However, one of the novelties of the current analysis is that we perform the analysis on sub-parts. This is because we are interested in knowing whether different functional categories differ in the extent to which they might be modular [ 1 ], not least because we also want to know whether this modularity might be reflected in such things as coexpression of the genes involved. This tendency is likely to vary by functional class. For example, cell-cycle genes should in principle show a strong coexpression signal if the modules are real. In contrast, one might imagine that all cell-signaling components need to be present under all circumstances and so coexpression need not be detectable. Analyzing the network as a whole, one might come to conclude that there exists no or just a weak correspondence between modules and coexpressed genes, when in reality there might be a very strong relationship for some categories while none for others. We therefore opted to analyze networks consisting of proteins belonging to different Munich Information Center for Protein Sequences (MIPS) protein functional categories [ 23 ]. This also has some methodological advantages. First, as methods for detecting protein-protein interactions may vary systematically according to functional grouping - for example, cytoplasmic complexes tend to be under-reported - it can be helpful to isolate each grouping alone. Second, it is probably desirable to filter out highly connected proteins to avoid big hubs and star-like clusters with low statistical significance [ 9 ]. Projecting the networks onto functional categories is a possible way of achieving such a filter. In every functional network, we found a regime of -values with significantly high average maximal overlap, that is, overlap equal to or greater than 0.8, and low ratios, characterizing the reliability of the proposed modular organizations. For an analysis of the performance of the algorithm as a function of -see Additional datafile 1. Note that these results extend the presence of modularity found previously in some yeast networks [ 9 , 10 , 24 ] to the functional networks introduced here. Explicit -values in the regime described above were chosen such that the average module size is around equal to 5 to 25 proteins, the so-called meso scale of biological networks [ 9 ] (Table 1 ). Modular phylogenetic profiles To ask whether the degree of phylogenetic correlation of the modules is higher than expected, we made use of the idea of phylogenetic profiles [ 18 ]; that is, patterns of presence or absence of homologs of a given protein across different genomes. We then adapted the underlying general assumption of phylogenetic profiles, that proteins belonging to a particular functional class should display a similar pattern of homologs in a set of organisms, to a more restricted hypothesis. We considered that modules within functional networks could indeed reflect a stronger functional link among their components than with the rest of the proteins. This stronger functional link, even when all proteins in the networks are part of the same functional classification, could consequently be reflected in the correlated presence or absence of module components across different organisms - that is, their phylogenetic profiles. To verify this initial suggestion, we examined the corresponding null hypothesis, that there is no phylogenetic correlation of the proposed structures, which is based on a completely uncorrelated distribution of phylogenetic profiles with respect to the modular organization. We made use of a class of statistical methods termed multi-response permutation procedure (MRPP). MRPPs are commonly used in ecological and environmental studies to compare an a priori group classification of a population in which measurements of r responses ( r ≥ 1) are obtained from each member of the population [ 25 ]. In contrast to well-known parametric statistical techniques such as the univariate and multivariate analysis of variance, MRPPs do not require any assumption with respect to the distribution of the response measurements. In the present case, proteins are the members of the population, modules are the group classification, and the phylogenetic profiles play the role of response measurements. A further difference from standard statistical techniques is that similarity measures, or normed distances, and not individual object measurements, are the primary units of analysis. We compared the within-module scores to the between-module scores. For each pair of modules we calculated each between-module protein pairwise similarity and took the average of these. To examine overall between-module similarity we calculated a weighted mean correlation of all between-module similarities. We then asked about the size of the difference between the mean within-module score and the mean between-module score, that is, ξ = - (see Materials and methods). Significance was tested by randomization; that is, we randomly permute the proteins within the modules while keeping the global modular organization fixed (Figure 1b ). Not all putative network modular organizations, according to different s, are shown to be biologically significant. However, we find for all networks a strong signal of phylogenetic correlation between genes in a module for some -values within the regime of high reliability of the algorithm (Table 1 and Additional datafile 1). We can extend the analysis to identify those modules showing the strongest signal. We used a method based on the analysis of each within-module similarity and the use of mean similarity dendrograms. For every module, we subtracted from the mean within-module similarity W m , the mean of all between-module similarity , a sort of representative of all pairs of between-module similarities: that is, ξ m = W m - . We estimated the significance of the values observed with such a modular test by performing again an approximate permutation procedure with a Holm's correction to multiple testing (Figure 1b and Materials and methods). This gives a significance measure of which module similarities reflect correlated evolution of their components in a particular functional network. Statistical significance does not supply any information on the magnitude of the respective similarities. To this end, we constructed a graphical representation, a mean similarity dendrogram [ 26 ], with branches for each module joined at a node plotted at . Branches terminate at W m , giving branch lengths of ξ m in similarity units (Figure 2 ). Those branches with considerable positive length, for example, ξ m equal to or greater than 0.1, indicate correlated evolution of the respective module components according to the phylogenetic profiles of the whole functional network, even though some of them could not be shown to be statistically significant because of the conservative nature of Holm's test. Thus, this combined approach provides both statistical significance and a clear quantitative picture of the compactness and isolation of the proposed modules. Figure 2 shows two examples of the application of this approach to evaluate modular network structures with the use of mean similarity dendrograms and phylogenetic profiles (we have chosen two small networks as examples to show a full picture of the modular characterization). Network phylogenetic profiles can be easily visualized as a matrix whose columns display the presence or absence of network nodes in a given organism and whose rows show the presence or absence of a given node in all the organism set. It then presents a full view of the degree of conservation of network modules for a collection of organisms. The arrangement of species in taxonomic groups is a convenient representation of the relative conservation of modules across the different lineages. Module cores Previous studies suggest that any given module may have a module core and a periphery [ 10 ]. In addition, in an evolutionary context, it is not clear to what extent full modules should be present or absent in different species, considering the tinkering aspect of most evolutionary processes. Can we use the network method to discriminate a core and does the core have a stronger phylogenetic correlation? To examine this hypothesis, we selected the most connected components of each module that was part of a given network, according to their intra-modular connectivity, and applied again the overall and modular tests to these cores (see Materials and methods). We found a substantial increase in the validation of the evolutionary significance of the modules revealed, for example, by the presence of a bigger number of significant modules (Table 1 , 'core' column group). Such statistically significant cores are mainly characterized by two distinct phylogenetic profiles; either their components had profiles with homologs present in all three kingdoms, or they had homologs present only in Eukarya (Table 2 ). This agrees with previous results and seems to support a picture of network assembly with a combination of ancient and modern modules [ 12 , 24 , 27 ]. The phylogenetic correlation suggests that this core architecture is biologically meaningful. Such extracted structures could then be used to probe this intermediate level of organization even in the case of uncharacterized biological systems. Owing to the extensive biochemical knowledge about yeast we are ready to validate such hypothesis. We have made use of the MIPs yeast complexes database [ 12 , 24 ] to characterize the biological relevance of the cores (see Additional data file 1 for a full list of phylogenetically distinct module cores and their biological characterization). As suggested, many, but not all, of the cores describe a significant part of relevant protein complexes, for example, anaphase-promoting complex, prenyltransferases (Ftase, GGTase I and GGTase II), some cytoplasmic translation initiation complexes such as eIF2 and eIF2B, Kel1p/Kel2p complex and Gim complexes (Table 3 ). Other module cores are not identified as parts of known protein complexes. This could mean either that some of the cores correspond to uncharacterized complexes or that these cores represent dynamic modules. Dynamic modules control a particular cellular activity by means of interactions of different proteins at different times or places instead of by the assembly of a macromolecular machine [ 1 ]. Thus, the combination of modular analysis and phylogenetic correlation is useful to find relevant components of biological systems. Do we also find that the significantly phylogenetically correlated cores have other properties of biologically relevant cores, that is, show a high degree of coexpression? We examined both the extent of coexpression [ 28 ] and degree of similarity in 5' motifs [ 29 ], the latter being an indirect method of assaying possible expression parameters. As regards coexpression, most functional groups have cores with more similar coexpression than expected by chance, but the significance levels tend to be low and hence the effect, while widespread, is relatively weak. This is probably a consequence of the dynamic organization of modularity [ 15 ], a phenomenon previously observed in protein complexes [ 28 ] (Table 4 and Materials and methods). This weakness is similarly reflected in the extent of sharing of 5' motifs. This latter result is probably as expected, given a lack of certainty over the relevance of many motifs and the fact that two genes of similar expression profile can have different motifs. Do the modules also represent units of homogeneity of dispensability? That is, if one protein in the core is lethal are all lethal, if one is dispensable are all dispensable? This can be quantified by the absolute distance of the ratio of lethal proteins in the core (0 ≤ ratio ≤1) to 1/2. We then sum these distances for the relevant cores in each network and estimate statistical significance by randomization (Figure 1b ). We find some cases where there is indeed higher homogeneity than expected (Table 4 ). But does this also mean that the modules all contain more lethals than expected? We find that for some functional groups this is indeed very profoundly the case. However, for other functional groups this is not so (Table 4 ). Assuming that the putative functional group of a protein can be assigned blind to genes, this method then has the potential to narrow down the possible drug targets in poorly described species. Perhaps as expected, cell-cycle, protein synthesis and transcription-related modules have the most significant tendency to amass lethal genes. Could we apply the knowledge of validated network structures in a therapeutical context, for instance to identify targets for antimicrobials? In principle, identifying candidate proteins as antimicrobial targets is straightforward: the protein needs to be in the microbe and not the host and to be essential to the microbe. To this end, we calculated the probability of finding lethal genes in the set of proteins without human homolog belonging to the significant cores. We compared this with the probability of finding lethal genes in those yeast proteins not found in humans which are part of the full network. While the data on which genes are essential is questionable, owing to condition-dependent lethality [ 30 ], the ratio of these two measures should give an indication of the extent to which our method improves the search strategy. Crucially, the method greatly increases the probability of finding such essential genes (Table 4 ). Some of these targets in yeast could be, for instance, the proteins APC4, ORC6 or POP5, which are part of complexes involved in the functional categories mentioned earlier (see Additional data file 1 for a detailed list). Conclusions We have shown that by combining protein-protein data and phylogenetic information it is possible to systematically describe biologically relevant modules in protein networks which partially correlate with other types of organization. The analysis also suggests, however, that not all core modules within the functional network are equally vital for the organism's survival. This may just reflect condition-dependent lethality [ 30 ]. Indeed, the fact that fewer than half of the core metabolic modules show significant enrichment for lethal genes is possibly due to such condition-dependency. Given this result, in the development of antimicrobials it seems wiser to attack modules related to transcription, protein synthesis and the cell cycle than it is to attack metabolic pathways. This simple example hints at the relevance of knowledge about the modular organization of networks in other therapeutic settings, such as that in cancer, to home in on which modules and which parts of modules within these systems should be selected in a putative list of potential drug candidates. Overall, our results contribute to validate the relevance of the modular level of organization of biochemical networks. Materials and methods Data We used two databases as of July 2003: MIPS [ 23 ], contributing 9,036 protein interactions; and DIP [ 31 ], contributing 15,116 interactions. Networks were assembled using a joint set of interactions after filtering common pairs. Protein information for the fully sequenced organisms selected is available at the website of the European Bioinformatics Institute [ 32 ]. A dataset on the presence of 5' regulatory motifs was downloaded from the Church Laboratory [ 33 ]. Expression data was obtained from a whole-genome mRNA expression data compiled by the Eisen laboratory [ 34 ]. Network clustering matrices Network clustering can be based on a global property, that is, L -based clustering, where L is referred to the shortest path length between two nodes in the network. From the interaction network, a matrix of distances is computed and transformed into an 'association' matrix by taking 1/ L 2 [ 10 ]. A second approach to network clustering is based on a local property, C -based clustering, where C is a generalized local connectivity coefficient measuring common interactors of any two proteins in the interaction graph [ 8 , 9 , 11 ] given by Here \|...\| denotes the size of the set, ∩ the intersection and Adj ( i ) the adjacency matrix, that is, the set of proteins interacting with protein i . Local properties tend to be more robust [ 11 ]. Module overlap Given two different modules, M i , M j , we considered the following overlap [ 13 ]: with \|...\| denoting the size of the set and ∩ the intersection. The average overlap used to determine the number of branches present in the clustering tree ( ) is given by: In this case, \| C \| and \| L \| denote the number of C -based and L -based modules extracted in a given functional network. Network small-worldness To characterize the small-world property of the networks, we first calculated the clustering coefficient, , and characteristic path length, L , for all assembled networks. = 2 j / m ( m - 1), the ratio between the number of interactions found among the m proteins connected to a given one, say j , and the maximal potential number of such interactions, which equals m ( m - 1)/2 for a undirected graph. We obtained high values of such clustering coefficient and small characteristic path length for all cases, reflecting the small-worldness of the networks. To assess the statistical significance of these values, we generated 100 randomly rewired graphs for each functional network with the algorithm described in [ 21 ]. All cases were shown to be highly significant ( P = 0.01), that is, , and L ≥ L random (we obtained P < 0.05 for L in the case of the energy network). Phylogenetic profiles We calculated binary and continuous phylogenetic profiles [ 18 ] for different threshold values, obtaining robust results for all discussed tests in both cases. For each yeast protein of interest, BLAST searches were done against 70 proteomes of species from the Archaea (14), Bacteria (47), and Eukarya (9) (see organism list in Additional data file 1). BLAST hits with Karlin-Altschul E -values bigger than a given threshold, E th , were considered absent [ 35 ]. A particular value is then assigned to each homolog present, characterizing in this way every protein by means of a phylogenetic vector. For continuous profiles, homologs receive a score of -1/log E and the absent ones receive a score of -1/log E th . For the binary case, profiles take the value 1 or 0 when the E -values are below or above the threshold, respectively. Finally, note that E -values were corrected to account for the different database sizes. Results in the main text are for the case of binary phylogenetic profiles and a threshold value of E th = 1 e -6 . Multi-response permutation procedures Non-parametric randomization methods, such as MRPP, have several advantages compared to more well-known parametric procedures. In particular, if the assumption of normally distributed populations is not reasonable, the datasets have multiple measurements and if multivariate comparisons are desired [ 25 ]. Similarity measure Given two binary phylogenetic profiles corresponding to proteins i , and j , we considered the following matching coefficient as a simple similarity measure: S ij = ( x + w )/( x + y + z + w ), where x is the number of homologs present in both phylogenetic profiles, y is the number present in profile i only and z is the number present in profile j only. Finally, w is the number of absent homologs in both profiles. Mean within and between similarities Within similarity Here, c m is the ratio between the number of components of module m , n m , and the number of components of all modules, N M , that is, c m = n m / N M , W m is the mean of similarities between proteins belonging to module m , and M is the total number of modules. Between similarity: Here, c m,s is the ratio between the product of the number of components of modules m and s , n m- n s , and the total number of components squared, N 2 M , that is c m,s = n m n s / N 2 M . W m,s is the mean of similarities between proteins of modules m and s , and M is the total number of modules. Results for all discussed tests were robust to the use of Euclidean distances with continuous profiles instead of similarities with binary profiles, as it is argued in the main text. Holm's test The Holm test [ 36 ] is a method that gets round the problem of the Bonferroni procedure being too conservative, by means of the added power of sequential stepping versions of the traditional Bonferroni tests. The procedure behind the Holm test is to find all the P -values for a set of k individual tests that are being performed and then rank them from smallest to largest. While Bonferroni would compare all null hypothesis to the same value α , the Holm test compares the smallest to α / k and, in case of rejection of the null case, to decreasing values α /( k - 1),... until failing to reject the null. To perform the MRPP Holm test, we computed the branch length, that is, W m - (see above) and determined the unadjusted P -value for each module by means of a permutation test with 10,000 randomizations. Suppose that we have M modules. We assemble an ordered vector of size M whose components are the uncorrected P -values in increasing order, that is, P 1 is the smallest uncorrected P -value and P M is the largest. To adjust a particular vector component P i we multiply this component by A i = ( M - i + 1), thus generating a vector P for adjusted P -values. The added power of the Holm test can then be seen in a simple example. Imagine the case of three modules, that is, M = 3. The uncorrected P -values of the corresponding MRPP tests are: P ν = (0.01, 0.02, 0.03). A Bonferroni procedure for multiple testing would consider only the first test as significant according to a 0.05 significancy threshold. However, the adjusted P -values obtained with the Holm test would imply that all tests are significant, that is, = P ν × (3,2,1) = (0.03, 0.04, 0.04). Core components To obtain the core component of the modules, we selected for each module those components with more than two interactions, for the case of a module whose component with maximal number of interactions (MNI) is less than ten, or those components with more than four interactions for the case of a module whose component with MNI is equal to or greater than 10. Slight modifications to these rules produced similar results. 5' regulatory motifs, coexpression and lethality of module cores For each of the significant module cores, ξ m ≥ 0.1, we calculated the mean of pairwise Euclidean distances between expression vectors of proteins belonging to a given module core. In the case of the 5' motifs, the statistic measures the number of regulatory motifs common to at least more than half of the core size. Finally, for each significant core, we simply measured the number of components that are lethal. The overall statistic for all cases is the sum of each corresponding measure in each core weighted by the ratio of the core size vs network size. P values are obtained with 10,000 randomizations. Additional data files Additional data file 1 , available with the online versin of this article, includes a discussion on the network clustering algorithm, the list of species and lineages for the phylogenetic profiles, and a list of phylogenetically distinct module core components and their biological characterization. Supplementary Material Additional data file 1 A discussion on the network clustering algorithm, the list of species and lineages for the phylogenetic profiles, and a list of phylogenetically distinct module core components and their biological characterization Click here for additional data file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545784.xml
554104	Khat use as risk factor for psychotic disorders: A cross-sectional and case-control study in Somalia	Background Little is known about the prevalence of khat-induced psychotic disorders in East African countries, where the chewing of khat leaves is common. Its main psycho-active component cathinone produces effects similar to those of amphetamine. We aimed to explore the prevalence of psychotic disorders among the general population and the association between khat use and psychotic symptoms. Methods In an epidemiological household assessment in the city of Hargeisa, North-West Somalia, trained local interviewers screened 4,854 randomly selected persons from among the general population for disability due to severe mental problems. The identified cases were interviewed based on a structured interview and compared to healthy matched controls. Psychotic symptoms were assessed using the items of the WHO Composite International Diagnostic Interview and quantified with the Positive and Negative Symptoms Scale. Statistical testing included Student's t-test and ANOVA. Results Local interviewers found that rates of severe disability due to mental disorders were 8.4% among males (above the age of 12) and differed according to war experiences (no war experience: 3.2%; civilian war survivors: 8.0%; ex-combatants: 15.9%). The clinical interview verified that in 83% of positive screening cases psychotic symptoms were the most prominent manifestations of psychiatric illness. On average, cases with psychotic symptoms had started to use khat earlier in life than matched controls and had been using khat 8.6 years before positive symptoms emerged. In most cases with psychotic symptoms, a pattern of binge use (> two 'bundles' per day) preceded the onset of psychotic symptoms, in contrast to controls of the same age. We found significant correlations between variables of khat consumption and clinical scales (0.35 to 0.50; p < 0.05), and between the age of onset of khat chewing and symptom onset (0.70; p <0.001). Conclusion Evidence indicates a relationship between the consumption of khat and the onset of psychotic symptoms among the male population, whereby not the khat intake per se but rather early onset and excessive khat chewing seemed to be related to psychotic symptoms. The khat problem must be addressed by means other than prohibition, given the widespread use and its role in Somali culture.	Background The present study investigated the relationship between psychotic symptoms and the abuse of khat in the Horn of Africa. The leaves of the khat shrub ( catha edulis ) are traditionally chewed in Arab countries, the Horn of Africa and East Africa [ 1 ] and recently this habit has spread to Western countries including the USA [ 2 ]. Due to improved transportation facilities, khat consumption has substantially increased during recent decades. This is reflected in the most recent issue of the World Drug Report: in 2001 five countries reported an increase in khat use and none a decrease; in 2002 an increase was reported in four, a decrease, again, in none [ 3 ]. Kalix (1996) [ 4 ] estimates that about 6 million individual portions are consumed each day worldwide. During our field work in the city of Hargeisa, North-West Somalia (Somaliland), where khat use is not restricted by law, we observed that current ways of intake do not correspond to the documented traditional use in the region. The traditional way of consumption was socially highly regulated: adult males (more seldom females) would gather and chew khat together at a so-called 'khat party', usually on weekends and afternoons until the time of the evening prayer [ 5 , 6 ]. Contrary to this formerly restricted use, current habits involve use by adolescents, chewing khat in tea-shops that operate day and night, early morning use, as well as "binging" and "speed runs" that may last for more than 24 hours. Our study was based on observations by social workers of collaborating non-governmental organizations and by our team during field studies on war-related trauma. It was shown that the widespread use of khat is related to the large number of individuals with visible signs of psychosis who are either homeless or kept in hiding, e.g. in physical chains, by family members who afraid to expose them to the general public. The main psycho-active component of khat leaves is cathinone (S(-)alpha-aminopropiophenone) [ 7 ]. Cathinone resembles amphetamine in chemical structure and affects the central and peripheral nervous system [ 8 , 9 ] and behavior [ 10 , 11 ] similarly. Amphetamines and some of its derivatives have been shown to induce psychotic symptoms in experimental settings in humans [ 12 , 13 ] and animals [ 14 ] and have been known to exacerbate psychotic states in psychiatric patients [ 15 , 16 ]. Similarly, khat-induced psychotic states have been described in several case studies [ 17 - 20 ]. However, the number of group, community and population-based studies on khat use and psychiatric symptoms [ 21 - 23 ] is still limited. Despite the ongoing scientific debate about amphetamine-induced psychosis it remains unclear whether the use of amphetamine-like substances including khat may actually cause a psychotic disorder in an otherwise healthy individual, or trigger the onset of schizophrenia in an individual with high vulnerability to the disease [ 24 , 25 ]. The fact that presumed amphetamine psychoses do not fully remit within weeks of abstinence in a substantial percentage of individuals [ 26 ] may also suggest that those individuals actually had a amphetamine-triggered schizophrenia. Increased drug use among psychotic patients may also come from an attempt to counteract nonspecific physical symptoms or side effects of neuroleptics [ 27 ]. The first goal of this study was to verify the impression of an unusually high prevalence of psychotic disorders in the city of Hargeisa via an epidemiological survey carried out in a representative sample of households. In addition, we wished to study the association between khat abuse and psychotic disorders. If khat abuse does induce psychotic disorders, a higher prevalence of psychotic diagnoses, mainly in men (as women rarely consume khat), was to be expected in Hargeisa compared to localities with less khat use. In addition, a case-control study served to examine whether individuals identified in the survey suffering from psychotic symptoms presented a pattern of khat consumption different from matched controls. Methods Sample In order to screen for households with mentally ill members, 612 households with 4,854 members were randomly selected as representative of the city of Hargeisa (approximately 400,000 inhabitants). For household selection the town was first subdivided into 30 sections of approximately equal populations. For this purpose we used the same sections as UNICEF in their assessment of vaccination coverage for children in October 2002, which were defined in a joint approach by UNICEF and the municipality of Hargeisa. A section had to be subdivided into square-shaped clusters if it was L-shaped, had a natural boundary within its limits (e.g. a steep hill) or had a much greater length than depth. A map for each section or cluster was produced using an aerial photograph. For random selection the geographical center of each cluster was determined and a random direction was chosen using a compass, a watch and a list of random numbers between 1 and 12 (a watch was oriented according to the compass, whereby 12 o'clock was adjusted to North; the random number defined the direction according to the hours on the face of the clock). Following the random direction until the border of the cluster was reached, all houses on the right side within a range of 15 meters were numbered. The first household to be approached was selected by a second set of random numbers. The next houses were selected by door-to-door distance until the predefined number was reached. Trained local staff interviewed the heads of the 612 households. If the head of household was not available, another adult member of the household was asked to answer to the questions. If no household member could be interviewed, the next house was selected according to the selection procedure. The following question was used to determine whether a person severely disabled due to mental health problems resided in the household: "Are there any members of your household who currently have mental problems that are so severe that the person has been unable to provide income or has been unable to help in the household for at least four weeks?" This criterion was fulfilled for 169 (137 male, 32 female) cases. These individuals will be referred to as 'positive screening cases'. A subsample of 52 'positive screening cases' was then randomly selected and examined in a clinical interview. In this group there were 44 males and 8 females. These individuals will be referred to as 'examined cases'. For each 'examined case' a matched control, who did not meet the criteria for disability due to mental health problems, was identified. Controls were matched by gender, age, and educational level. These 'case controls' were subjected to the same clinical interview as the examined cases. Forty-nine of the 52 examined cases and one control were diagnosed with psychiatric or neurological disorders (on this basis we estimated a sensitivity of 0.98 and a specificity of 0.94 for the screening procedure). We included only those forty-three examined cases (82.7%) in the further analysis who – in addition to the impairment of function – showed as main manifestations of mental problems at least one very severe or two moderate positive psychotic symptoms in the absence of organic somatic damages that might explain them; from hereon these are termed 'cases with psychotic symptoms'. The disorders of other examined cases were stroke (2), traumatic brain injury (1), mental retardation (2), and dementia (1). The case control with a positive diagnosis reported hallucinations with intact reality testing during khat intoxication and was replaced by a healthy individual. Socio-demographic characteristics for the two studied groups are summarized in Tables 1 and 2 . Table 1 Socio-demographic characteristics of the sample of N = 4,854. For socio-demographic data, mean ± standard deviation and percentages and absolute numbers (in parenthesis) respectively are reported. P-values refer to differences between ex-combatants, civilian war survivors, and persons without any war experience tested by Kruskal-Wallis 1 and chi 2 2 . Whole group Ex-combatants Civilian war survivors No war experience p Total Number 4,854 250 2,667 1,937 - Male 2,449 240 1,201 1,008 Female 2,405 10 1,466 929 < 0.001 2 Age 22.2 ± 17.8 42.8 ± 13.5 28.7 ± 16.6 10.7 ± 12.1 < 0.001 1 Age range 0 – 102 12 – 102 0 – 101 0 – 90 - Years of formal education 2.6 ± 4.1 5.2 ± 5.6 3.3 ± 4.3 1.4 ± 3.0 < 0.001 1 Economic situation of household* 2.8 ± 1.8 2.5 ± 1.7 2.9 ± 1.7 2.8 ± 1.8 0.005 1 Percentage of khat users in week before interview among men > 12 yrs 31.3% (495 of 1,581) 60.3% (144 of 239) 28.1% (306 of 1,090) 17.9% (45 of 252) < 0.001 2 Khat bundles/week before interview among khat chewing men > 12 yrs 7.6 ± 4.4 7.9 ± 4.8 7.6 ± 4.4 6.7 ± 3.2 0.544 1 'Screening cases' among men > 12 yrs 8.4% (133 of 1,581) 15.9% (38 of 239) 8.0% (87 of 1,090) 3.2% (8 of 252) < 0.001 2 'Screening cases' among women > 12 yrs 1.9% (30 of 1,600) 20% (1 of 5) 1.6% (22 of 1,372) 2.8% (7 of 248) 0.066 2 * The household economic situation was approximated as the sum of four significant assets (water tap, electrical power, TV set, car) and type of housing (hut = 1, house = 2, closed compound = 3). The mean of the two ratings is presented. For the combined rating Cronbach's Alpha = 0.78; correlation with income = 0.73, p < 0.001. Table 2 Socio-demographic characteristics of the 43 cases with psychotic symptoms and 43 'case controls.' Mean ± standard deviation and percentages and absolute numbers (in parenthesis) respectively are reported. P-values refer to group differences assessed by t-test/Wilcoxon test. Patients with psychotic symptoms Matched controls Sig. total male female total male female p N 43 38 5 43 38 5 1.0 age 33.9 ± 10.5 34.6 ± 10.0 28.6 ± 13.8 34.7 ± 10.3 35.3 ± 10.0 30.4 ± 12.8 0.718 Education (yrs in school) 5.6 ± 4.5 6.4 ± 4.3 0.0 6.3 ± 4.9 7.1 ± 4.6 0.0 0.511 Martial status (% married) 16.3% (7) 18.4 % (7) 0 % (0) 44.2% (19) 50.0% (19) 0% (0) 0.005 Employed or at school 0% (0) 0% (0) 0% (0) 41.9% (18) 47.4% (18) 0% (0) < 0.001 Procedures and materials All interviews were carried out in October and November 2002. Prior to the interview, participants were informed about the background of the study and the survey procedure. All participants signed a written informed consent, which was first read and explained to them (as most of the participants were illiterate). In the case of mentally challenged participants, background and procedure were also explained to the responsible caretakers whose written informed consent was a prerequisite for their participation. Interviews were approved by the local authorities and the National Demobilization Committee of Somaliland. Representatives of the different communities were informed and invited to observe the assessment in the field. Newspaper advertisements, daily radio programs and flyers informed the population about and helped them to understand the real purpose of the assessment (at first rumors had spread that the research team would provide free medication for mentally disturbed individuals and created unreal expectations). Consequently, the level of cooperation was extremely high. Out of 73 households approached under the supervision of the first author only three refused their participation. Therefore, we estimate that in total less than 5% of all households refused participation. Local interviewers were recruited among NGO and hospital staff who already had experience in working with mentally disabled persons. They participated in a two-week training course, which involved teaching the basic psychiatric concepts (e.g. psychotic symptoms), interviewing skills, training on the screening-instrument and field work under close supervision of experts. After the end of the course, fourteen of the seventeen trainees were employed for the duration of the study. Five interviewer teams, each comprising one male and one female interviewer, and four local supervisors did the field work. The local supervisors received additional training on the random sampling method. During the first weeks of interviewing the first author closely supervised the teams in the field. The screening interview assessed individual socio-demographic information, khat consumption and experiences in the civil war (subjects were grouped as either active war participants, civilian war survivors, or without any war experiences). Khat consumption (number of bundles/week) was assessed for the week prior to the interview. The household economic situation was approximated as the sum of four significant assets (water tap, electrical power, TV set, car) and type of housing (Table 1 ). Clinical interviews were carried out by some of the authors (MO, MS, CC, BL), all trained in the assessment of psychotic disorders and PANSS rating. Socio-demographic information and war-history were detailed. A standard event list was used to quantify the number of traumatic event types a person had experienced; the list included 11 types of events: active combat experience, accident, natural disaster, witness murder or killing, rape, sexual molestation, physical assault, being kidnapped or captured, torture, other, or suffered shock because a close person had experienced a traumatic event. In a short semi-structured qualitative interview the main areas of psychological condition and functioning were assessed. Additionally, psychotic symptoms were assessed using the items of the Composite International Diagnostic Interview (CIDI), Section G [ 28 ]. The individual's age at which the family first noticed positive psychotic symptoms that prevailed over six months was taken as indication of the onset of a psychotic disorder. Khat consumption (average number of bundles/day) was assessed for the week before the interview and for the week prior to the onset of symptoms in examined cases. Case controls were asked about consumption in the week prior to the interview and for consumption at the age of symptom onset reported by his/her examined case-pair (Table 2 ). As many examined cases were not able to give valid information, the primary care taker and other family members were also interviewed. Clinical interviews were administered in the English language with the help of trained local interpreters, who had participated in the same training course as the interviewers. After the interview, the interviewing psychologist rated the current psychopathology using the Positive and Negative Syndrome Scale, PANSS [ 29 ]. All items of the standardized interviews were translated from English into Af Somali by mixed teams of clinical experts and bilingual staff. Several steps of independent back-translations and corrections were necessary until a satisfactory translation was achieved. Content validity was assured by the involvement of clinical experts at all levels of the translation process. Also, interpreters were introduced to the concepts targeted by the respective questions. Data analysis Differences between cases and controls were confirmed using chi 2 -tests (or Fisher's exact tests), binomial testing, Student's t-test or Wilcoxon, and ANOVA or Krsucal-Wallis test (all tests two-tailed). Univariate Analysis of Variance (ANOVA) was used to explore the effects of war-trauma on the khat intake. Means and standard deviations are reported. Results Of the sample of 4,584 inhabitants of Hargeisa the screening interview disclosed 169 positive screening cases (i.e. 3.5%). Positive screening cases were more often male than female (133 of 1,581 men, i.e. 8.4%, and 30 of 1,600 women above the age of twelve years, i.e. 1.9%; p < 0.001; Table 1 ). Khat chewing was more frequent among male positive than among negative screening cases above the age of 12 years: 46.6% of the 133 positive screening cases had chewed khat in the week before the interview in contrast to 29.9% of the 1,448 negative screening cases (p < 0.001). Consumers among positive screening cases had also chewed a greater amount of khat in the week preceding the interview (positive screening cases: 4.1 ± 6.3 bundles; negative screening cases: 2.2 ± 4.0 bundles; p = 0.001). The proportion of positive screening cases was substantially higher among males above the age of 12 who had active war experience (ex-combatants) than in male civilian war survivors of the same age (p < 0.001, Table 1 ). The latter proportion was significantly higher than in men without any war experience (p = 0.007). Psychotic symptoms meeting our criteria were determined for 83% (43) of the examined cases. Retrospective investigation suggested that the onset of psychotic signs occurred at the age of 23.4 ± 9.9 years (N = 38 men: 24.1 ± 9.8 years; N = 5 women: 18.4 ± 9.6 years; p = 0.230). PANSS ratings of these 43 cases showed a substantially higher magnitude of current psychotic symptoms compared to a sample of 240 medicated schizophrenic patients [ 30 ] in the following subscales: Positive 27.1 ± 7.3, Negative 32.0 ± 8.9, Composite -4.9 ± 10.6, General Psychopathology 52.7 ± 12.7, Anergia 16.6 ± 5.5, Thought Disturbance 16.4 ± 5.3, Activation 7.8 ± 3.0, Paranoia/Belligerance 10.8 ± 4.1, Depression 9.5 ± 3.9, and Supplemental Scale 17.4 ± 7.4. In further exploratory observations, we noticed a high tendency towards aggressive and hyperactive behavior. During interviews, most patients reported that they were in contact with a ghost ('djin'), often associated with auditory, visual and somatosensory hallucinations. Fifteen of the 43 cases with psychotic symptoms (35%) were under current medication at the time of assessment; another nine (21%) had received medication in the past. A variety of drugs had been given, ranging from neuroleptics (12 patients) to prometazine (6), benzodiazepines (3), tricyclic antidepressants (3), carbamazepine (1) and other unknown drugs (10). Uncontrollable (disruptive, violent) behavior had led to restraint of cases with psychotic symptoms by chaining them to an object in 28 of the 38 men (i.e. 74%) and 3 of the 5 women (i.e. 60%) (p = 0.608). Additionally, 9 men (24%) and 3 women (60%) had been locked up to control their behavior (p = 0.589). On average, the 31 cases with psychotic symptoms who had ever been chained had spent 4.2 ± 5.2 years in chains (men: 4.5 ± 5.4 years; women: 1.8 ± 1.9 years; p = 0.410) and the 10 cases who had ever been locked up spent on average additional 5.2 ± 6.5 years restrained (men: 4.0 ± 6.6 years; women: 10.0 ± 4.2 years; p = 0.264). The proportion of khat user was higher among cases with psychotic symptoms than among matched controls: all except one of the 38 male psychotic cases, in contrast to 25 of the 38 male controls, had used khat (p < 0.001). In the cases with psychotic symptoms, regular khat consumption had started at an earlier age (16.6 ± 4.8 years) than among their matched controls (20.7 ± 7.0 years, p = 0.010; Figure 2 ). All except one case with psychotic symptoms (compared to 61% of controls, i.e. 14 of 23) had started to chew before the age of 23 years (p = 0.004). None of the women admitted to having ever chewed. Figure 2 Khat intake and traumatic experiences. Average daily khat intake in bundles (at the age when the case with psychotic symptoms showed onset of symptoms) according to number of traumatic life events. We divided the cases with psychotic symptoms and case controls into three groups according to the number of event types experienced as follows: none or one: 11 cases and 13 controls; two or three: 9 cases and 15 controls; more than three: 11 cases and 14 controls. Bars indicate mean and standard error. In the weeks preceding the onset of psychotic symptoms, cases with psychotic symptoms had chewed an average of 2.5 ± 2.0 bundles/day compared to 0.5 ± 0.6 bundles/day in controls (p < 0.001; Figure 1 ). Excessive khat intake (> two bundles/day) in this period was found in 78% of chewers among male cases with psychotic symptoms (i.e. 29 of 37) but in only 4% of chewers among controls (i.e. 1 of 25; p < 0.001). For the cases with psychotic symptoms, the age of onset of khat chewing correlated significantly with symptom onset (r = 0.70, p < 0.001). The lapse of time between first use of khat and onset of symptoms was greater than one year in 31 of the 38 male cases with psychotic symptoms (i.e. 82%); it varied around a mean of 8.6 ± 6.6 years (median 7 yrs). In the week before the interview 54% of the lifetime khat chewers among male cases with psychotic symptoms (i.e. 20 of 37) and 36% of the controls (i.e. 9 of 25) had chewed khat (p = 0.162). Furthermore, psychotic patients consumed an average of 1.5 ± 1.0 bundles/day, with controls consuming an average of 0.9 ± 0.7 (p = 0.172; Figure 1 ). Figure 1 Patterns of khat consumption. Left: age of first khat intake among lifetime chewers in patients with psychotic symptoms (35) and case controls (23); right: amount of khat use (in 'bundles' per day) in the week before the onset of first positive symptoms (25 cases with psychotic symptoms, 24 controls) and in the week before the clinical interview (16 cases with psychotic symptoms, 9 controls). Bars indicate mean and standard error. Khat use correlated with severity of symptoms: In cases with psychotic symptoms onset of khat use correlated significantly with higher scores on the PANSS subscales Negative Symptoms (r = - 0.36, p = 0.039, N = 34), General Psychopathology (r = - 0.44, p = 0.009; N = 34), Anergia (r = - 0.39, p = 0.022, N = 34) and Thought Disorder (r = - 0.42, p = 0.014, N = 34), while the amount of khat consumed prior to onset of psychotic symptoms correlated significantly with the subscale Paranoia/Belligerence (r = 0.49, p = 0.006, N = 30) and the Supplemental scale (r = 0.50, p = 0.005, N = 30). The amount of khat consumed in the week before the interview correlated with the PANSS subscale Anergia (r = - 0.35, p= 0.029, N = 38). The number of traumatic event types experienced did not differ between cases with psychotic symptoms and matched controls (cases with psychotic symptoms: 3.1 ± 2.3 events; matched controls: 2.9 ± 2.4 events; p = 0.749). Also, the age when the first traumatic event was experienced did not differ between them (cases with psychotic symptoms: 20.2 ± 9.6 years; matched controls: 20.6 ± 9.2 years; p = 0.853). In order to estimate the relationship between number of experienced traumatic events and khat intake we used univariate ANOVA with khat intake before onset of psychotic symptoms (for controls at the age of symptom onset of matched pair) as dependent variable, and number of traumatic events in three categories (no or one event, two or three, four or more) and being a case or a control as independent variable. We found significant effects for the number of traumatic events (Sum of squares 36.6; df 2; F = 17.028; p < 0.001) and for the variable 'case or control' (Sum of squares 53.9; df 1; F = 50.136; p. < 0.001) and a significant effect of their interaction (Sum of squares 31.0; df 2; F = 14.404; p < 0.001) (total R 2 = 0.620). In Figure 2 the results are displayed graphically. Discussion The present study revealed a number of interesting and relevant findings. (1) In a representative subsample of male inhabitants of Hargeisa (older than 12 years) we found 8.4% severely disabled due to mental problems; of these, 83% had severe psychotic symptoms. Compared to the prevalence rates of psychotic disorders reported for male samples elsewhere [ 31 , 32 ] this is higher than expected. Furthermore the gender ratio among the mostly psychotic positive screening cases was unusual (1 women to 7.7 men). (2) Khat abuse showed a significantly earlier onset and was more frequently excessive in male cases displaying psychotic symptoms than in matched controls. (3) There is a higher vulnerability to disability due to mental disorders in those groups of society who were directly involved in combat in the past. (4) Among the positive screening cases examined and their matched controls we found that khat intake before the onset of psychotic symptoms (respectively at the same age for matched controls) was higher when more traumatic events had previously been experienced. These results are intriguing, as we may have detected only the very tip of the iceberg with our sub-optimal screening procedure, leaving less severe cases of mental and neurological disorders undiscovered. The findings of the present study suggest that there is an association between khat consumption and psychosis; however the existence of a causative relationship and its direction between the two remains unclear. Furthermore, it seems that it is not the consumption of khat per se but rather specific patterns of it that are related to psychosis, especially early onset in life and the excessive intake (> two bundles per day). Both seem to be related to active participation in war, e.g. child ex-combatants used khat while fighting in order to counteract fear and enhance performance [ 33 ]. Excessive khat consumption has already been reported to be associated with higher severity of psychiatric symptoms [ 21 ]. Our data also suggest that there might be a moderating effect of the number of traumatic experiences on subsequent khat intake. This would be in line with the self-medication hypothesis. Khat consumption might be related to the outbreak of psychosis in various ways. Considering psychoses as the result of genetic and acquired vulnerability and additional stress factors, the early onset of khat abuse may have substantially increased the risk in already vulnerable individuals. In animal studies, damage imposed on the developing brain, e.g. by drugs, increases the potential of amphetamine-like agents to change neuro-chemical systems and to induce psychotic-like behavior [ 34 ]. In humans, drug abuse during puberty has been found to precede the onset of psychotic symptoms and prodromi [ 35 ] and to be related to poorer treatment outcome [ 36 ]. Additional risk factors and particular stressors, such as war experiences, may contribute to an increased vulnerability. It is presently unclear, however, whether traumatic experiences act indirectly through higher drug consumption in traumatized men or whether they exert a direct effect on the brain, increasing the risk of developing psychosis. The temporarily higher khat abuse preceding first symptoms in cases with psychotic symptoms compared to case-controls may suggest that khat consumption is the primary agent, causing the onset of psychoses. This assumption is further strengthened by the inequality in the gender ratio, as women are generally denied access to the drug. Moreover, khat consumption may affect the course of a psychotic disorder and the maintenance of symptoms. In contrast to the high khat consumption prior to the onset of psychotic symptoms, the amount of current khat consumption did not differ between cases with psychotic symptoms and controls and a significant number of patients did not have current access to the drug. Drug-effects on the course of illness may be attributed to sensitization [ 37 ] or to lasting neurotoxic effects of prolonged stimulant intake [ 14 ]. To clarify the question of how continued khat consumption might affect the further development of psychotic symptoms, the number of relapses related to khat intake would be of tremendous importance. Our observation was that once a patient has developed severe psychotic symptoms he is restrained (e.g. chained) and kept away from the drug until symptoms remit. However, as soon as he is released the patient engages again in (often excessive) khat consumption until symptoms relapse and he is restrained again. Many patients had lived through this circle several times. In Western samples of amphetamine-induced psychosis, first relapses have mostly been studied [ 37 ]. Furthermore, it seems plausible that the conditions under which cases with psychotic symptoms were frequently kept may have infuenced the course of any psychopathological development. Some methodological shortcomings of the study are related to the nature of field work in this specific post-conflict setting, which involved restricted freedom of movement due to the security situation and limited resources. However, although a perfect design is not possible in a rather complex field situation, the importance of such studies is evident from the fact that little or no information from post-war Somalia is available. First, we could not control whether our sample was comparable to the population from which it was drawn due to the lack of recent statistics in Somalia. Second, the validity of our screening and clinical interview can be questioned because of various points, e.g. whether the descriptions of symptoms and disability might be adequate for the Somali culture, or whether factors such as interpretation or the interviewing of a whole family rather than a single patient might distort the information. In our approach we decided to use disability as a selection criterion and a descriptive rather than a normative approach to identify the reasons for it. Thus, we decided to use the more unspecific terms 'cases with psychotic symptoms' or 'psychotic symptoms' rather than 'schizophrenia' or 'drug-induced psychosis'. However, the fact that our local interviewers found 123 of the 169 positive screening cases (72.8%) restrained, i.e. chained or locked up, and the overall high average PANSS scores of the examined cases, show that there are 'hard' criteria, which justify the use of the term 'psychosis' and validate our screening. Nevertheless, we recognize that with our method we must have failed to discover persons suffering from psychotic and other mental disorders (false positives). But assuming that this error is somehow the same for the whole sample, our finding that the group of society that engages most in khat chewing (males above the age of 12) most often suffers severe impairment due to mental disorders shows evidence that khat is related to mental health problems. Thirdly, the fact that we could not choose our matched controls from the pool of negative screening cases – the resources that would have been necessary to (repeatedly) contact and arrange appointments with them exceeded our capacities – leads without any doubt to an overestimation of the specifity of our detection procedure. Furthermore, the retrospective third-person assessment of the onset of psychotic signs, as well as the retrospective and subjective assessment of khat use, will have introduced some error variance in the data. However, we believe that especially in the Somali culture khat intake cannot be compared to any food intake, of which the retrospective assessment is problematic. In the Somali culture khat has a special significance, which also comes from existing traditional knowledge about its dangers. Therefore we suppose that retrospective assessment of khat intake is more valid than that of food intake. Also, the effects of social desirability might have affected both the detection of positive screening cases and the assessment of khat use. The fact that dozens of families approached the compound of our collaborating partner agencies in order to find help for their mentally ill members (often they brought them in chains to the compound) shows the great need for mental health services. Thus, the tendency to hide family members from our interviewer teams can be estimated as marginal. On the contrary, during the interviews our greater fear was that households would over-report the presence of mentally challenged family members. For this reason, we counted only those persons as positive screening cases when their existence in the household could be validated (e.g. by their physical presence). The assessment of khat consumption is another point where effects of social desirability can be assumed. Whereas the information given by examined cases could be validated by the information of family members, we were well aware of the danger that the matched controls might have under-reported their khat intake. In order to allow for this in the interview we spent much time and effort to identify the real khat intake (e.g. often we had to 'negotiate' about the answer to the khat questions – a habit inherent in Somali culture). Nevertheless, the high correlations validate the assessment procedures. Also, the sample size was too small to answer some of the questions in detail, especially the small number of female examined cases. However, the results were nonetheless statistically very robust. In sum, our design could not determine the existence of a causal relationship between khat and psychosis – this would have been too ambitious a goal. However, our main findings do not contradict the hypothesis that khat might cause psychosis. Future research should focus on the question of causality. Unfortunately, the Horn of Africa is probably the region where stimulant abuse currently reaches highest levels on a world-wide scale. This circumstance offers a unique research opportunity for cross-sectional and repeated measurement studies, which would enable us better to understand the relationship between schizophrenia and drug effects and to fill the gap in knowledge related to khat use. At the same time, the alarmingly high prevalence of khat chewing among persons who suffer from psychotic symptoms in one of the countries of its highest use, and the magnitude of human suffering associated with it, demands the development of adequate community-based prevention and treatment interventions. The usefulness of standard procedures derived from developed countries and brought to the Horn of Africa must be viewed in a "service-research attitude"[ 38 ]. We believe that khat abuse has become a tragic obstacle for the reconstruction of this war-torn society; consequently, there is an urgent need to address this mental health issue with means other than prohibition and regulation of the demand side through taxation, as khat is integral to the Somali culture. Conclusion Not khat intake per se but rather specific patterns of it are linked to the development of psychotic symptoms, like early onset in life, excessive chewing (> 2 bundles/day) and use as self-medication for war trauma related symptoms. Although we cannot establish a causal relationship between khat and psychosis, we find some evidence that the prevalence of psychotic disorders is increased among the male adult population of Hargeisa. Ex-combatants are the group in society who are most affected by severe mental disorders, among them we find the highest abuse of khat. The way of khat use in Hargeisa is changing, indicated by e.g. the development of a group of heavy users who show consumption patterns similar to amphetamine addicts in Western countries. Measures of prevention and treatment of and further research on khat-related severe mental disorders have to be undertaken, however, taking into consideration that khat is an integral part of the Somali culture, which cannot simply be prohibited. Competing interests The author(s) declare that they have no competing interests. Authors' contributions Development of the study design and selection of instruments were accomplished by MO, FN, TE, MS, BR and HeH. MS, TE and MO performed pilot studies. Training and supervision of local interviewers was provided by MO, HaH, FN and BL. Clinical interviews were conducted by MO, MS, CC and BL. Statistical analysis was performed by MO and TE. The article was composed and revised by MO, TE, BR, FN and HeH. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554104.xml
548277	Reflections of physiotherapy students in the United Arab Emirates during their clinical placements: A qualitative study	Background Although Western models of education are being used to establish health professional programs in non-Western countries, little is known about how students in these countries perceive their learning experiences. The purpose of this qualitative study was to describe the reflections of physiotherapy students from a Middle East culture during their clinical placements and to compare them to reflections of physiotherapy students from a Western culture. Methods Subjects were six senior students (3 females, 3 males, mean age 22.6 years) and 15 junior, female students (mean age 20.1 years) in the baccalaureate physiotherapy program at a university in the United Arab Emirates (UAE). They wrote weekly entries in a journal while in their clinical placements. They described an event, their reaction to it, and how it might affect their future behavior. Two evaluators independently read and coded the content of all the journals, and then worked together to categorize the data and develop themes. A third evaluator, an UAE national, independently read the journals to validate the content analysis. A feedback session with students was used to further validate the data interpretation. The themes were compared to those derived from a similar study of Canadian physiotherapy students. Results The content of the students' reflections were grouped into 4 themes: professional behavior, awareness of learning, self-development and shift to a patient orientation, and identification and analysis of ethical issues. Although the events were different, students from the UAE considered many of the same issues reflected on by Canadian students. Conclusion Physiotherapy students from a Middle East culture consider many of the same issues as students from a Western culture when asked to reflect on their clinical experience. They reflect on their personal growth, on how they learn in a clinical setting, and on the ethical and professional behaviors of themselves and others.	Background Reflection is considered by many [ 1 - 3 ] in the Western world to be an important part of clinical practice and clinical decision-making. In order to learn from experience, health care professionals must reflect on the events and determine where the information 'fits' in relation to their assumptions, previous experience and knowledge. Reflection is the process where individuals think about and evaluate their experience in order to come to new understandings and appreciations [ 2 - 4 ]. All promoters of reflection refer to it as a process of thinking about experiences. The process leads to a change in perspective or understanding. Schön [ 2 , 3 ] suggested that reflective practitioners are better able to manage the uncertainty and complexity of clinical practice, and therefore asserted that a major part of preparation of professionals should be centred on enhancing their ability to reflect. Journal writing is a method that has been used to promote reflective thinking in health science students and professionals [ 5 , 6 ], to facilitate the link between academic learning and clinical practice [ 7 ], and to assist students to explore and change their attitudes toward patients [ 7 , 8 ]. When writing a reflective journal, students are asked to describe a learning event or situation, explain how the experience led to new understandings and appreciations, and consider how they might act differently in the future. Students have indicated that the writing of the journal provides a framework for the reflective process).)[ 9 , 10 ]. Shepard and Jensen [ 11 ] specifically referred to the need for physiotherapists to have 'reflective' knowledge in order to deal with uncertainties and value conflicts. Two recent Canadian studies [ 12 , 13 ]. have described the reflections of physiotherapy students during clinical placement. The students wrote weekly in journals, and reflected on a number of topics that were classified into six major themes [ 12 ]. These included: 1) the process of making clinical decisions; 2) the complexity and richness of interactions with clients; 3) the influence of the practice environment on learning and patient care; 4) acquisition of clinical and administrative skills; 5) the value of clinical experiences in validating and integrating previous learning; and 6) acknowledgment and evaluation of different learning methods. Students frequently commented on their own values and beliefs and whether these were in harmony or conflict with the beliefs and actions of other health professionals or clients [ 13 ]. They discussed professional behavior, professional collegiality, respect for others, advocacy and informed consent. In considering the student reflections, the investigators wondered about the potential impact of culture and working environment on the content of the reflective journals. Would students from a non-Western culture and a different health care system be concerned about the same issues? Would they feel comfortable with the reflective process, particularly documenting their thoughts in a journal? How might the hospital/institutional infrastructure and norms affect their learning? We had a unique opportunity to answer some of these questions by comparing the themes from reflective journals of physiotherapy students in the United Arab Emirates (UAE) with those taken from the above mentioned studies [ 12 , 13 ]. conducted at McMaster University in Canada. The physiotherapy program at the University of Sharjah in the UAE was developed with a philosophy similar to that of the program at McMaster, and the Chair of the program was a McMaster faculty member. Students in both programs have worked in small groups, studied similar content, and considered the same professional and attitudinal issues such as moral reasoning, client-centred care, professional behavior and professional code of ethics. Although both the UAE and Canada have many of the conveniences of modern society, there are major societal and cultural differences. The students in both countries have similar standards of living and have ready access to education and health services. Many of the health problems of modern society – diabetes, heart disease, arthritis, cancer, and trauma from traffic accidents – are common in Canada and the UAE. However, Canada is a democracy with a predominantly Christian and European population, whereas the UAE is a system of emirates with a population comprising mainly Arabic and Eastern cultures and where the Muslim religion predominates. The students in the two countries might be expected to have different values and beliefs, and different societal norms that could affect their reflections. The purpose of this study was to describe the reflections of physiotherapy students from the University of Sharjah during their clinical placements. A second objective was to compare the themes generated from the Sharjah data to the themes from the previous two studies on reflections of McMaster students during their clinical placements. Because the students in the two programs have similar socioeconomic status and similar curricula, it was anticipated that differences in reflections would be due to differences in culture and life experiences related to the cultures. Methods This qualitative study was conducted during the students'clinical placements in a summer semester of the Physiotherapy Program at the University of Sharjah, United Arab Emirates, a program developed in affiliation with McMaster University, Hamilton, Ontario, Canada. Curriculum and semester description The baccalaureate physiotherapy program in the UAE comprises one pre-professional year (year 1) and three professional years (years 2–4) of study. To be admitted to the program, students must have completed the UAE Secondary School Scientific Certificate (equivalent to grade 12), or equivalent program accredited by the Ministry of Education. They must have also scored at least 500 on the Test of English as a Foreign Language (TOEFL). While in the physiotherapy program, students need to complete university required and elective courses. Basic science courses are offered in years 1 and 2. Physiotherapy courses in years 2–4 include small-group, problem-based learning tutorials and clinical skills laboratories based on problems/ case scenarios enhanced with resource sessions. The male and female students are taught separately in discrete areas of the campus. Students Six senior physiotherapy students (3 females and 3 males) with a mean age of 22.6 years (range 20.5 to 23.1) and 15 junior, female students with a mean age of 20.1 years (range 19.9 to 22.8) formed the subject group of this study. These students were respectively from the first and second cohorts of students admitted to the new physiotherapy program at the University of Sharjah. All students spoke Arabic and English. The senior students had completed the academic component of year 3 and were in their third clinical placement, which provided them with six weeks experience primarily with clients with cardio-respiratory conditions. The junior students had just completed two academic semesters of year 2, and were in their first clinical placement – four weeks with clients with peripheral musculoskeletal conditions. Students practiced in five different clinical sites in the UAE, under the supervision of clinical preceptors and university instructors/coordinators. The study was approved by the Ethics Committee of the College of Health Sciences, and all participating students signed an informed consent. Evaluators Two instructors were involved in reading, coding and identifying the major themes that emerged from the students' reflective journals. One instructor (HL) from the University of Sharjah, Department of Physiotherapy, had taught one course to the junior group and two courses to the senior group. The second instructor (JW) from McMaster University Physiotherapy Program, had taught one course to the senior group as a consultant to the program. The two instructors were not involved with the students during their summer clinical placements. A third evaluator (AAS) independently read all the journals. She was an instructor in the Sharjah program, a UAE national, and fluent in both Arabic and English. She had taught two courses to the junior students. Her analysis was used to confirm the interpretation of the entries made by the other two evaluators who were both from Western cultures. Procedure The students were asked to write a reflective journal, a new exercise for all students. This assignment was one of the requirements for a Pass mark in their summer clinical placement. The students were required to make one entry per week for the duration of their clinical placement (four weeks for junior students and six weeks for senior students), and to submit the journal weekly to the university instructor involved in their clinical supervision. However, the instructor did not provide any feedback to the student at that time. The students were instructed to include the following in their reflective journals: Reflective journals should include observations, impressions, and reactions to what you have learned in the academic portion of the semester and how you are applying it to clinical practice. How does the clinical experience change what you thought, felt, or did in the past, and how you may respond in the future? You are expected to write at least one journal entry per week during your clinical placement . 1. Describe the learning event, issue or situation. Describe prior knowledge, feelings or attitudes with new knowledge, feelings or attitudes. What happened? 2. Analyse the learning event, issue or situation in relation to prior knowledge, feelings or attitudes. What was your reaction to the learning event, issue or situation? Your response may include cognitive and emotional reactions. Why did it happen? 3. Verify the learning event, issue or situation in relation to prior knowledge, feelings or attitudes. What is the value of the learning event, issue or situation that has occurred? Is the new knowledge, feeling or attitude about the learning event, issue or situation correct? 4. Gain a new understanding of the learning event, issue or situation. What is your new understanding of the learning event, issue or situation? 5. Indicate how the new learning event, issue or situation will affect future behavior. Determine the clarification of an issue, the development of a skill or the resolution of a problem. How will you approach the same or similar event, issue or situation in the future? Data analysis The method described by Coffey and Atkinson [ 14 ] was used to analyse and interpret the data in the students' journals. The analysis was conducted in two phases after the students had completed their clinical placements and submitted their entire journal. In Phase 1, one evaluator (HL) selected five journal entries based on their readability level and diverse content and forwarded them to the second evaluator (JW). The two evaluators read and coded the five journals independently. Then they met to discuss and establish agreement on the coding. In Phase 2, the two evaluators independently read and coded all remaining journals. They met again to 1) determine agreement on the content of each entry; 2) group the codes into categories; and 3) determine major themes. When coding discrepancy occurred, the evaluators discussed the discrepancy and came to a consensus. The following two methods were used to further validate the analysis and interpretation of the data. 1) The third evaluator independently read and labelled all journal entries to confirm the interpretation of the content. 2) The themes established by the evaluators were presented to two students who had completed the journals, and who volunteered to provide feedback on the authors' interpretation of their reflections. Themes for the present study were developed without direct reference to the themes found in the Canadian studies [ 12 , 13 ]. The Emirati and Canadian themes were then compared by examining their descriptions and content. Results The two initial evaluators (HL, JW) came to a consensus on four themes concerning the clinical learning experience of the students. These themes did not change as a result of the coding of the third reader (AAS) or the feedback from the students. However, these additional data occasionally resulted in a change in emphasis of some of the content within a theme. Whenever this happened, the differences are described. All students outlined the positive impact and value of their learning from the clinical placement. They discussed events and issues that fit into the following four themes: 1. Professional behaviors: skills and attitudes, scope of practice, time management, professional boundaries, and respect for clients and colleagues. 2. Awareness of learning: clinical versus academic learning, gaining of important new knowledge, self-directed and life-long learning, and client as a source of learning. 3. Self-development and shift to a more client-oriented focus over the time of the clinical placement. 4. Identification and analysis of ethical issues. Each of these themes is discussed below with quotes provided for illustrative purposes. Professional behaviors The students appeared very aware of their own behavior and that of other health professionals and students. They reflected on how they should behave as professionals, but this also included how they should behave in general. The events that precipitated discussion on this theme included actions of themselves and other health professionals and included what they regarded as both positive and negative behaviors. They referred to physiotherapists and other health professionals who helped the students and who modelled good professional behavior. They also described some situations where health professionals did not show sufficient respect for their clients or their colleagues, or did not provide 'best practice'. They commented on professional boundaries, being concerned about the client-therapist relationship becoming too personal and limits to the scope of physiotherapy practice. In addition, the students noted that self-directed learning and life-long learning were professional responsibilities. This topic is discussed under the next theme, Awareness of Learning. The students providing feedback emphasized that professional behavior (both negative and positive) was also learned from patients who might show their appreciation, be patient or uncooperative, or provide specific feedback regarding the student's communication or skills. The students also provided examples of "crossing boundaries" in terms of professional competencies and relationships with their patients and supervising therapists. One patient asked me angrily where [were the physiotherapists]? I responded politely to wait until they finish their work with the patients... I calmed her down and asked her to be patient...I really felt that the [physiotherapist] and I should maintain and manage our time...It is really a big responsibility we have . [student 11, week 3] The child came crying and refusing to look at her forearm. We should respect the emotional status of the child and try to understand it because the child has been through a lot. The accident..., surgery,...and functional limitations...The stress should be controlled before giving physiotherapy...I should be patient and try to give the most enjoyable set of exercises to gain the child's trust . [student 10, week 3] Sometimes patients are shy to tell that there is no benefit from the treatment programme. So I asked the patient if he feels any improvements and if not to inform me in order to change the plan of treatment. The patient responded very well to that . [student 5, week 3] Awareness of learning Although the entire reflective journal assignment was designed to have students reflect on their learning, this theme was included to capture the many components of learning discussed by the students. They gained knowledge, but also commented on the unique aspects of clinical learning. Clinical experiences provided an opportunity to apply and enhance their academic learning. Junior students particularly noted how observing surgery helped them understand their anatomy, or how working with "real patients' was not as straight forward as their academic learning. The senior students realized the need to be life-long and self-directed learners because they were always encountering new conditions/situations and they wanted to provide their clients with 'best practice'. Students acknowledged the clients as a source of knowledge, and noted that clinical situations provided a stimulus to learn. In the feedback session, students particularly emphasized how the clinical experience had enhanced their learning "a lot!" They also indicated that clinicians reinforced the need for life-long learning because of the constant changes in science. Some therapists welcomed new knowledge from students, for example, learning about outcome measures, while others indicated they did not have the time. It was really good to have a mix of people [clinicians] and to learn from their experience in the work field...Each one of them has given us a new way to deal with patients and... also a new experience to add to our future knowledge... The learning is not finished...We have to keep our hands on the best information available and the best experience . [student 4, week 6] [I] try to apply the theoretical bases learned at the university. [I] interact with patients and take their histories, try to know their goals and what they need from physiotherapy. I try to invent new materials for the exercises. In my opinion, when we apply what we learned in the theory, the information sticks in our minds and is hardly forgotten . [student 21, week 2] Self-development and orientation shift The theme of self-development was derived from the content of individual student entries, but was also seen in the change in the reflections as students proceeded through their placements. Often at the beginning of the clinical encounter, the students were unsure of themselves, but they gained confidence as they worked with the client and/or learned from their colleagues. The change across the journal entries was even more noticeable. In many of the early entries, students described their own emotional response to a client or a situation. They were concerned about their own performance, and about what they were learning from the placement. They indicated how an event had an impact on them. The later entries were more client-centred. The focus was more on how the client responded to an event, or how helpful a physiotherapist was. Students also referred to an increased confidence, the more experiences they had. It became evident from the third reader's summary that students were aware of how their professional behavior, outside direct patient care, could ultimately have an impact on the patient. They referred specifically to the maintenance of knowledge and skills and to relationships with other health professionals. Students in the feedback session referred to their initial lack of confidence which improved during the clinical placement. They added, however, that it was difficult to demonstrate the "correct confidence level" to their instructor/evaluator, particularly when students were occasionally told they were "over-confident". They felt that self-development was affected by behavior of other physiotherapy students, method of feedback from instructors and whether their clients improved or not. The quote immediately below illustrates how a student confronted his/her uncertainties during one client encounter. The next quote concerns a student's acknowledgement of the change in his/her confidence with increasing experience. First patient with tetraplegia...What if I do something wrong?...First, worried... and unable to do anything, I observed other colleagues... I could do it too! [student 6, week 2] A one-month old infant was in the ICU. I thought I would not be able to touch her as usual, but things are now different. I did all the treatment... with my hands with no hesitation and I was not scared... My reaction was totally changed and it is becoming more regular... I am happy... I trust myself better and it is increasing with time...and will increase the more cases I face . [student 3, week 6] In the quotes below, the student initially talks about his/her own emotional response to a patient, but later in the placement discusses the need to adapt to patients' situations and provide them with the necessary treatment. In both journal entries, the student discusses a client with a novel and somewhat "unpleasant" condition. However, the student reacts differently in week 5 than week 1. At that moment I was shocked because I was not familiar with the neurology cases. When the physiotherapist asked me to help...I hesitated in the beginning. ...This situation had a bad effect on me ... I was depressed and I couldn't do any single movement [student 4, week 1] I have seen a patient who was always under sedation due to his pain and his anger which was there almost all the time. ... I was always confused ...not knowing what to do for him and what is the best for him. ... Such a case made me think [very] much before any and each simple movement. ... So this made me prevent [other] problems such as ulcers, and invent any simple movement just to change his lying position [student 4, week 5] As the quotes illustrate, the student was concerned about what to do in both cases, but by week 5 was able to overcome the emotional response, problem solve about the management of the client, and take action. Ethics Students commented on what they believed to be ethical or unethical behavior of the health professionals they were in contact with. They noted when informed consent was not obtained from clients and they discussed ways to get consent from uncooperative clients. They talked about having respect for their clients, and maintaining confidentiality. They noted "ethical" and "unethical" behavior in the same professionals under different circumstances. The staff is cooperative and helpful, respect each other, but there is no confidentiality or respect of time with patients . [student 20, week 1] They were concerned when individuals (clients or professionals) were being treated unfairly or differently because of their health condition or socio-economic status. In some cases they thought that the client might be harmed by the practice. One student was concerned about how to 'tell the truth' to a client who had a poor surgical result possibly due to the questionable practice of the surgeon. One student described an event where a therapist refused to see a patient when she changed her appointment time, but treated another patient who did not have an appointment. The student felt that the latter patient was treated because she was a "VIP (very important person)". I felt sad for the [person who did not receive treatment], and I said to myself, where are the ethics? I learned before ... that all patients should be at the same level without any bias. ...all persons who work in health care fields specially the professional therapists must [follow] the code of ethics because they have humans in their hands. ... I will delete the word VIP from my life work . [student 12, week 2] In the feedback session, the students also indicated how clients could be discriminated against because of their health condition and the therapist's level of confidence. Therapists might provide less treatment to a child if they felt uncomfortable treating children. They might leave a student to treat an older person if they felt the management of older persons was less important or less effective. Feedback from students The students agreed with the four themes and did not think that any major concept was omitted in this analysis. However, they added some insight to the content and interpretation of each theme. The students emphasized that many people affect their learning – university instructors, therapists, other health professionals, students and patients/clients. They felt that the information derived from the analysis of the reflective journals was very important. They strongly recommended that the results be disseminated to clinicians, instructors and future students to increase their awareness of the learning process and help the students to improve. Comparisons of themes of students from McMaster University and the University of Sharjah The content of the journals indicated that students from both universities were comfortable with the reflective process. They frequently shared quite personal information, including their positive and negative emotions, concerns about their own behavior, and a discussion of their own beliefs. Some students in both countries, simply described an event and provided minimal personal reflection, but these students were in the minority. Table 1 summarizes the comparable themes of the Emirati and Canadian students. Themes from both countries related to professional behaviors, learning and ethical issues. Self-development was not identified per se as a theme in the Canadian studies, but change and growth were mentioned under the other themes. Table 1 Themes from Emirati and Canadian students during clinical placements UAE Canada Professional behaviors † Process of making clinical decisions † Complexity and richness of interactions with patients ‡ Respect: for uniqueness of individual ‡ Professionalism: responsibility and behavior as a member of a profession ‡ Professional collegiality: interaction with and respect for health professionals including physical therapists Awareness of learning † Effects of practice environment on learning and patient care † Acknowledgment and evaluation of different learning methods † Value of clinical experiences in validating and integrating previous learning † Acquisition of knowledge and clinical and administrative skills Identification and analysis of ethical issues ‡ Allocation of resources ‡ Advocacy: for clients, society or health policy ‡ Informed consent: right of person to decide what will happen to him/her † Themes from Williams et al [12] ‡ Themes from Geddes et al [13] In the area of professional behavior, students in both countries commented on role models (both positive and negative) in the clinical environment, as well as on their own behavior or response to clients. Both groups demonstrated a respect for their clients and saw their role as going beyond physical treatment. Canadian students mentioned advocacy in the community while students in the UAE discussed supporting their clients within the hospital milieu. Both groups talked about the uniqueness of learning in clinical placements. The clinical experience enhanced and confirmed their academic learning, and stimulated them to learn more. They enjoyed learning new information and using their knowledge to help others. They were anxious when they "didn't know what to do", but expressed increased confidence with each experience. They appreciated constructive feedback on their performance from clinical supervisors, health professionals and clients. Both groups acquired new skills related to direct client care (therapeutic techniques and communication) and administrative activities (time management, organizational skills and charting). Only Canadian students referred to writing reports and billing, tasks that students in the UAE were not expected to do in their clinical placements. Both groups, however, broadened their view of the scope of practice of physiotherapy. Students from both countries noted ethical issues pertaining to inequalities in their respective health care systems. Canadian students were concerned about the shortfall of resources (time, personnel, funding), and about differences in health care provided to private clients or those funded through provincial health insurance. Emirati students noted that the health or societal status of a client could affect their treatment within the health system. Discussion Physiotherapy students from Sharjah demonstrated the ability to reflect on their clinical practice at the highest level (discussion of how experience will impact on future behavior). They described not only the learning event, but also their emotional and cognitive reactions to it. They analysed the situations to arrive at new understandings and considerations of how they would behave differently in the future. Like the students in the Canadian studies, the subjects in the present study described three types of learning that are considered essential for clinical decision-making. These are propositional (academic learning – facts, concepts), professional craft (learning from experience), and personal knowledge (knowledge of self and unique frame of reference) [ 15 ]. The students commented on the knowledge that they gained (e.g., anatomy, treatment techniques, medications), and the unique learning opportunities of fieldwork (e.g., application and enhancement of academic learning, learning new skills, response to novel situations). They acknowledged that their personal frame of reference affected their clinical interactions, but was also modified by their clinical experience. Although the students did not categorize their learning in the three areas, they were able to describe the acquisition of knowledge and skills required for making clinical decisions. The reflection on one's own development is a common theme in the reflections of health professionals. Jensen and Denton[ 7 ] referred to themes of student self-adequacy and inadequacy in their study of 23 physiotherapy students completing journals in their first clinical placement. Tryssenaar and Perkins[ 16 ] demonstrated that reflection on personal growth was still evident at the end of the educational programs and the beginning of careers for physical and occupational therapists. Subjects in their study described initial anxiety and then increasing confidence about their ability as graduate therapists. They became more patient-centred with experience. Jensen and Paschal[ 17 ] suggest that the shift to a client-centred approach is part of the transition that students go through from their first to later clinical placements. Because we did not follow one group of students over several placements, we cannot comment on the change in the focus of students with each additional clinical experience. However, both junior and senior students described their personal reactions more frequently at the beginning of the placement, and the patient's unique situation more frequently in the later weeks. Perhaps with each new experience (e.g., start of a clinical placement) students will be temporarily more self-centred as they deal with their anxieties and uncertainties. As they become more comfortable with their own abilities, they can focus more on the client. Such a pattern is in agreement with the finding mentioned above, that therapists become more client-centred with experience. [ 16 ] One other study [ 18 ] examined the reflections of non-Western physiotherapy students during their clinical placements. Although the students were practicing in an English, Western environment, rather than in their own culture and language, the students and their supervisors did raise some issues that may have relevance to the present study. The Chinese students and their Australian supervisors noted that the students were reluctant to self-evaluate, to express their opinions, to admit they did not understand or to take active steps to acquire needed information. In the present study, the students certainly voiced their opinions, admitted their perceived inadequacies, and expressed pride in their achievements in the reflective journals. However, some may have been less forthcoming with their clinical preceptors or university instructors/coordinators, particularly when they did not agree with them. In the sample quotes in the Results section, the students did not mention discussing their concerns and feelings with their supervisors. On the other hand, the students in the present study were working in their own language and culture, and would be better able to express their views, and to do it in a "politically correct" manner. Unlike the Chinese students, the students in the UAE discussed the need for self-direction in their learning. This difference could be cultural and/or due to the self-directed nature of the problem-based learning method used in the physiotherapy program at the University of Sharjah. Language might have been a factor in the reflections of the students from the UAE, however. Although the students worked in Arabic in the clinical environment, they conducted their physiotherapy education and wrote their journals in English. It is possible that some students had difficulty accurately expressing their reflections in English, and that there was some misinterpretation by the evaluators. However, two of the evaluators (HL, AAS) knew the students well and were used to their writing. One of them (AAS) was a UAE national and fluent in both English and Arabic. When the two initial evaluators (HL, JW) worked together in Phase 1 to determine coding methods, part of the discussion was on how to interpret the "second-language writing" of the journals. In spite of these precautions, it is still possible that the themes may have varied somewhat if the journals had been written in Arabic and interpreted by Arabic-speaking evaluators. Conclusions Physiotherapy students from a Middle East culture consider many of the same issues as students from a Western culture when asked to reflect on their clinical experience. They reflect on their personal growth, on how they learn in a clinical setting, and on the ethical and professional behaviors of themselves and others. Competing interests The author(s) declare that they have no competing interests. Authors' contributions HL and JW were involved in the conception and design of the study, in the qualitative analysis, and in the writing of the manuscript. HL coordinated the data collection. AAS assisted with data analysis. All authors were involved in the preparation of the manuscript, and read and approved the final version. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548277.xml
555540	Clinical outcome of endonasal KTP laser assisted dacryocystorhinostomy	Background To evaluate the clinical outcome of primary endonasal laser assisted dacryocystorhinostomy (ENL-DCR) using the potassium-titanyl-phosphate laser. Methods We retrospectively reviewed all primary ENL-DCRs performed within a period of twelve months by the same combined Ophthalmology and Otorhinolaringology team in Freeman Hospital, Newcastle upon Tyne, UK. The main outcome measure for success was resolution or significant improvement of epiphora. Details of surgery, intraoperative and postoperative complications, as well as pathology associated with failure were also studied. Patients were followed up for at least 12 months. Results A total of 41 consecutive ENL-DCRs on 29 patients (22 females, 7 males, mean age 75 years) were analysed. All patients had bicanalicular silicone intubation for at least 4 months. The success rate at 12 months postoperatively was 78.1%. Pathology associated with failure included: intranasal pathology (12.2%), mucocele (7.3%), and systemic sarcoidosis (2.4%). No significant intra-operative complications were recorded. Conclusion The ENL-DCR with potassium-titanyl-phosphate laser can be considered as a safe and efficient primary procedure for the treatment of nasolacrimal duct obstruction.	Background Dacryocystorhinostomy (DCR) is the treatment of choice for patients with chronic stenosis and obstruction of the nasolacrimal duct. External dacryocystorhinostomy (EXT-DCR) was first described by Toti [ 1 , 2 ] in 1904. The endonasal approach was first introduced in 1893 by Caldwell [ 3 , 4 ], but it was inherently limited by poor visibility of endonasal anatomy during surgery. The introduction of high-resolution fiberoptic endoscopes in the late 1980s enabled adequate visualisation of the nasal cavities, and permitted minimally invasive surgery, under local anaesthesia, avoiding visible facial scarring[ 5 , 6 ]. Endonasal dacryocystorhinostomy (ENL-DCR) can be performed either entirely surgically[ 7 ] or with the assistance of laser to create the fistula. Massaro, Gonnering and Haris [ 8 , 9 ] were the first to describe the endonasal dacryocystorhinostomy (ENL-DCR), using Argon laser for the creation of the DCR fistula. Since then, carbon dioxide (CO 2 ), holmium:Yag (Ho:Yag), neodymium:Yag (Nd:Yag), and potassium-titanyl-phosphate (KTP) laser systems have been employed in an attempt to identify the optimal delivery system that would achieve sufficient bone ablation with effective haemostasis[ 10 ]. Reported primary ENL-DCR success rates vary from 68% to 99% [ 5 , 6 , 11 ], depending on the type of laser, the size of the osteotomy and the use of antimetabolites, such as mitomycin C [ 12 ]. In this study, we evaluate the clinical outcome of 41 consecutive primary ENL-DCRs that were performed on 29 patients using the KTP laser over a period of one year. Methods The records of all 47 ENL-DCR procedures that were performed in Freeman Hospital, Newcastle upon Tyne, United Kingdom, within a period of twelve months were retrospectively studied. The patients' main symptom was moderate to severe epiphora. Obstruction of the nasolacrimal system distal to the lacrimal sac was diagnosed with nasolacrimal syringing. Radiographic imaging was not part of the routine preoperative evaluation. Otorhinolaryngological preoperative assessment included full endoscopic examination of nasal cavities, looking for evidence of mucosal disease including polyps particularly in the middle meati. Exclusion criteria for ENL-DCR were: noticeable lower lid laxity, previous lacrimal surgery, suspicion of malignancy and previous radiation therapy. Each patient with primary nasolacrimal duct obstruction was counselled as to the advantages and disadvantages of EXT- DCR versus ENL-DCR, together with estimated success rates of the two different types of DCR. The operations were performed by the same ophthalmologist (C.N.) and ENT surgeon (S.C.). The majority of patients had surgery under local anaesthesia on an outpatient basis, except for one who opted for general anaesthesia. For local anaesthesia, Amethocaine drops were instilled in the conjunctival sac, followed by injection of Xylocaine 2% with 1:200000 Adrenaline in the medial third of both eyelids and transcaruncularly to the lacrimal sac. Cophenylcaine spray and intranasal cocaine 4% paste was applied to achieve anaesthesia and haemostasis. Dilatation of the lower punctum was performed and a 20G vitreoretinal probe was inserted in the lower canaliculus and advanced into the nasolacrimal sac. The light was directly visualised endonasally with a 0° rigid nasal endoscope, and the laser energy was delivered, with full laser precautions, via a KTP laser probe guided by the light. The nasal mucosa and lacrimal bone were ablated and the ostium was enlarged anteriorly as necessary with a microronguer. Bicanalicular O'Donoghue silicone tubes were inserted and secured with a Watzke sleeve. A course of topical Chloramphenicol drops was given for 1 week. Patients were examined 1 week postoperatively and then at 6 months for removal of tubes, or earlier, if discomfort was experienced. Mean follow up period was 16 months (range 12–24 months). Results Forty- seven consecutive primary ENL-DCR operations with lacrimal intubation were performed from March 2001 to February 2002 on 35 patients with primary acquired nasolacrimal duct obstruction. Five cases with incomplete follow up and one which had to be converted to EXT-DCR due to very thick lacrimal bone were excluded from the study. Forty-one ENL-DCR procedures on 29 patients (7 males and 22 females) were included in the study. Mean age was 75 years (range 47–90, SD 13.7). 12 patients (41.4%) underwent simultaneous bilateral surgery and 17 (58.6%) had unilateral ENL-DCR. In total, 19 right sided (46.3%) and 22 left sided (53.7%) procedures were recorded (Table 1 ). Pre-operatively, all patients were suffering from significant epiphora, which was affecting their quality of life. Mucocele was present in 6 cases (14.6%) and previous dacryocystitis in 4 (9.8%), while in another 3 cases (7.3%) these two conditions co-existed (Table 2 ). Mild medial ectropion was noted in 5 cases (12.1%) and intranasal pathology (including sinus disease, deviated nasal septum, polypoidal medial turbinate and previous nasal fractures) in 7 cases (17.1%). Two patients (4.8%) had systemic sarcoidosis. During the operation the mean laser energy used was 400.2 joules (range 96–797, SD 188.5). Serious intra-operative complications did not occur, although in some patients' records per-operative mild discomfort was documented. The removal of silicone tubes was scheduled at 6 months post-operatively, though in ten patients extubation was performed at 1–4 months due to discomfort. In two patients, mild nasal haemorrhage was noted on removal of tubes. The operation was defined as being successful if the patient was asymptomatic or if there was significant improvement of symptoms, not requiring any additional procedure. Success rate at 12 months postoperatively was 78.1%. No improvement of symptoms was noted in 8 patients (19.5%), while 1 patient reported worsening of epiphora (Table 3 ). Pathology associated with failure (9 cases – 21.9%) included: intranasal pathology (sinus disease, septum deviation, polypoidal medial turbinate and previous nasal fracture) in 5 cases (55.5%), mucocele in 3 cases (33.3%), and systemic sarcoidosis in 1 case (11.2%) (Table 4 ). EXT-DCR or treatment of the nasal pathology was offered to all patients with persistent epiphora. Discussion ENL-DCR is a well established surgical technique with some advantages compared to the conventional EXT-DCR. These include limitation of tissue injury to the discrete fistula site, avoidance of a skin incision, excellent haemostasis, the ability to perform a lacrimal bypass operation on an outpatient basis, quicker patient rehabilitation, decreased overall health care expense and patients' preference [ 8 , 13 ]. Main limitations of the technique are its steep learning curve, the higher equipment cost and its contraindication in cases of severe pre-existing nasal deformities or scarring and suspected lacrimal sac neoplasms [ 13 ]. ENL-DCR avoiding the use of laser is a well described procedure. Several surgical instruments have been employed to remove the bone overlying the lacrimal sac, including drills, osteotomes, curettes and rongeurs. Weidenbecher reported resolution or improvement of symptoms in 95% of the patients[ 6 ]. In another study of surgical ENL-DCR results, Sprekelsen achieved good results on 96% of the operations[ 5 ]. Both studies describe no major complications associated with the technique. Various types of laser (Argon, CO 2 , Ho:Yag, Nd:Yag), have been employed in an attempt to achieve better bone ablation and haemostasis. Massaro et al [ 8 ] and Christenbury [ 14 ] reported a 70% success rate using an argon blue laser, but they both encountered difficulties in creating an adequate osteotomy. A prospective randomised comparison of EXT-DCR and ENL-DCR with the CO2-Nd:Yag laser by Hartikainen et al [ 15 ] revealed far superior results of the external approach (91% success compared to 63% with the endonasal technique), admitting though that their ENL-DCR technique was possibly suboptimal. Szubin et al [ 10 ] achieved an impressive successs rate of 97% with the Ho:Yag laser. This laser seems to outperform the rest, delivering better haemostasis and ablation, but its cost is higher and it is not so diverse in its applications [ 10 ]. The KTP laser, already utilised by ENT surgeons in other procedures, offers excellent haemostasis but its ablating properties are relatively poor, thus requiring the use of a microrongeur if the underlying bone is thick [ 16 ]. Mirza et al [ 17 ] reported improvement of symptoms in 64% of patients by KTP laser ENL-DCR, rising to 82 % including revision procedures. Using the same type of laser, Reifler found 68% success rate in a retrospective study of 19 cases, with a longer follow up of 10–16 months [ 18 ]. Interestingly, though, he noted that the first 10 cases showed a success rate of only 50%, compared to 89% in the following 9 cases. This observation reflects the steep learning curve of this technique. Mickelson et al [ 19 ] reported a series of 19 patients with 100% success (follow up 5–25 months). Hofmann et al [ 2 ] performed ENL-DCR with KTP using miniendoscopes to visualise the exact site of obstruction, and achieved success rate of 83% at one year follow up. In our experience, the success rate of primary ENL-DCR using the KTP laser at 12 months was 78.1%. We defined success as complete resolution of epiphora or improvement of symptoms with no further procedure required, as this outcome carries the most significant implication on the patient's quality of life. The anatomic result was not evaluated at postoperative follow up, as the healed intranasal ostium size and patency do not always correlate with symptomatic relief. A significant number of patients have been reported to have symptoms in spite of a patent fistula (54% for EXT-DCR and 39% for ENL-DCR)[ 20 ], where in some cases, paradoxically, resolution of symptoms is achieved despite a negative fluorescein test [ 21 ]. As the aim of our study was to evaluate the results of primary procedures only, repeat procedures were excluded. The optimum duration of stent retention following DCR is controversial. In published series it varies from 4 weeks to 6 months, though there is some evidence that prolonged silicone intubation may increase the incidence of DCR failure by inciting a granulomatous reaction at the internal ostium, with subsequent stenosis [ 22 ]. All our patients underwent lacrimal intubation with silicone tubes, and in most cases the tubes were removed at 6 months postoperatively. Early extubation (1–4 months) was necessary in 10 cases due to discomfort, and the operation failed in 2 of them (20%). Thus, in our experience, early removal of silicone tubes was not associated with lower success rate. Reported pre-operative risk factors for ENL-DCR failure include pre-existing sinus disease, mucocele, nasal septum deviation, connective tissue diseases such as sarcoidosis, previous EXT-DCR, other nasal surgery, nasal fracture, and thickened lacrimal bone [ 23 ]. In our series, failure was associated mostly with intranasal pathology (sinus disease, septum deviation, polypoidal medial turbinate and previous nasal fracture), but also with mucocele and sarcoidosis (table 4 ). Radiographic imaging, particularly CT DCG or CT of nose and sinuses could be of potential help in clarifying the extent of concomitant sinus and nasal disease and increase our success rate. However, as this would add to the cost and the complexity of the preoperative assessment in a busy clinical setting [ 24 ], it was not part of our routine evaluation. The common outcome in the failed cases is blockage of the ostium due to cicatrisation, adhesions between the ostium and the medial turbinate, synechiae between the ostium and the septum, or granuloma formation within the ostium [ 22 ]. Opinions differ about size and location, with some surgeons favouring smaller ostium size at the lower thinner part of the lacrimal bone [ 25 ], while others recommend larger size and removal of the thicker frontal process of the maxilla [ 6 , 26 ]. The ideal technique is yet to be defined. It has been proposed that the thermal energy produced by the laser may lead to scarring and subsequent blockage of the ostium [ 10 ]. It has also been suggested that the adjunctive intra-operative application of Mitomycin C (MMC) can be considered in high risk cases or primary failures, as it appears to be safe and efficient in improving the patency rate [ 9 ]. ENL-DCR performed under local anaesthesia is reported to be generally well tolerated by the patients[ 27 ]. In our study, all operations were performed under local anaesthesia, except for one patient who opted for general anaesthesia. No significant discomfort was reported by any of our patients, which confirms the reported positive patients' views. Reported complications associated with ENL-DCR include per-operative or post-operative haemorrhage, punctal erosion related to silicone intubation, silicone tubing prolapse, canalicular obstruction, orbital fat herniation, orbital and subcutaneous emphysema, conjunctival fistula formation, retrobulbar haemorrhage, and transient medial rectus paresis (23). In our series, no severe per-operative or post-operative complications were encountered. The only documented complications were discomfort caused by the silicone tubes in 10 patients requiring early extubation, and mild, transient nasal haemorrhage during removal of the silicone tubes in 2 patients. In our experience, the use of KTP laser in ENL-DCR under local anaesthesia with the adjunctive use of an osteotome is a safe and efficient technique, with good results. The particular advantages of this laser are its superior haemostatic properties and its diversity, which reduces the cost of the operation, as it is already employed in other procedures by the ENT surgeons. Significant complications are not common with this technique. In cases of failure, revision ENL-DCR or EXT- DCR can be performed. ENL-DCR with KTP laser is routinely performed under local anaesthesia, thus avoiding the risks of general anaesthesia usually required for EXT-DCR [ 24 ] The operative time is also shorter compared to the EXT- DCR [ 16 ]. Conclusion ENL-DCR using KTP laser appears to be an efficient technique, with low complication rate and it is well tolerated by the patients. It still needs refinement in order to achieve the higher success rate of the EXT- DCR, which remains the gold standard method for the treatment of nasolacrimal duct obstruction. At present, we believe that patients should be involved in the decision on the type of operation, after comprehensive consultation on the advantages and disadvantages of each technique. Competing interests The author(s) declare that they have no competing interests. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555540.xml
516445	Performance of dye-affinity beads for aluminium removal in magnetically stabilized fluidized bed	Background Aluminum has recently been recognized as a causative agent in dialysis encephalopathy, osteodystrophy, and microcytic anemia occurring in patients with chronic renal failure who undergo long-term hemodialysis. Only a small amount of Al(III) in dialysis solutions may give rise to these disorders. Methods Magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads in the size range of 80–120 μm were produced by free radical co-polymerization of HEMA and ethylene dimethacrylate (EDMA) in the presence of magnetite particles (Fe 3 O 4 ). Then, metal complexing ligand alizarin yellow was covalently attached onto mPHEMA beads. Alizarin yellow loading was 208 μmol/g. These beads were used for the removal of Al(III) ions from tap and dialysis water in a magnetically stabilized fluidized bed. Results Al(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was observed at pH 5.0. Comparison of batch and magnetically stabilized fluidized bed (MSFB) maximum capacities determined using Langmuir isotherms showed that dynamic capacity (17.5 mg/g) was somewhat higher than the batch capacity (11.8 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands. Al(III) ions could be repeatedly adsorbed and desorbed with these beads without noticeable loss in their Al(III) adsorption capacity. Conclusions Adsorption of Al(III) demonstrate the affinity of magnetic dye-affinity beads. The MSFB experiments allowed us to conclude that this inexpensive sorbent system may be an important alternative to the existing adsorbents in the removal of aluminium.	Background About 8% of the Earth's crust is comprised of aluminium. This element is the most abundant metal naturally present in air, soil and water. Consequently, environmental exposure to aluminium is potentially possible. Its ingestion is unavoidable since aluminium compounds are added not only to most water supplies but also to many processed foods and medicines. Aluminium is a known neurotoxicant. It enters the brain, where it contributes to some neuro-degenerative diseases including dialysis encephalopathy, osteomalacia, osteodystrophy, in particular those related to dialysis treatment of uremic subjects [ 1 ]. Only a small amount of Al(III) ions in dialysis solutions may cause these disorders. Aluminium may contribute to Alzheimer's disease [ 2 ]. Aluminium is also able to give rise to toxicity in the bones and hematopoietic system in humans [ 3 ]. Positively charged aqua and hydroxy-monomeric forms have been found to be the most toxic species of aluminium to living organisms in the terrestrial and aquatic environments [ 4 ]. Generally, aluminium sulphate is used as a coagulant in the treatment of water to help the removal of suspended matter and highly coloured humic substances [ 5 ], thus reducing the dose of chlorine later required to ensure satisfactory microbiological quality. Hence, potable water often contains high aluminium levels of natural origin and/or from the water purification process [ 6 ]. The selective removal of aluminium ions have been extensively investigated by applying several techniques [ 7 - 9 ]. Among them, the use of specific polymeric adsorbents has been considered as one of the most promising techniques [ 10 , 11 ]. Specific adsorbents consist of a ligand (e.g., reactive textile dye, ion-exchange functional groups or chelating agents) which interacts with the metal ions specifically, and a carrier solid matrix. There have been several separation approaches performed under magnetic field [ 12 ]. The most well known technique is the magnetically stabilized fluidized bed. Magnetically stabilized fluidized bed exhibits combination of the best characteristics of both packed and fluidized bed. These include the efficient fluid-solid mass transfer properties, elimination of particle mixing, low pressure drop, high feed-stream solid tolerances, good fluid-solid contact, elimination of clogging and continuous countercurrent operation [ 13 ]. Especially, when dealing with highly viscous mediums contact with the magnetic adsorbent in a magnetically stabilized fluidized bed is desirable because of high convective transport rates. Recently, there has been increased interest in the use of magnetic adsorbents in biomolecule coupling and nucleic acid purification [ 14 ]. Magnetic adsorbents can be produced using inorganic materials or polymers. High mechanical resistance, insolubility and excellent shelf life make inorganic materials ideal as adsorbent. The main disadvantage of inorganic supports is their limited functional groups for ligand coupling. Magnetic adsorbents can be porous or non-porous [ 15 ]. They are more commonly manufactured from polymers since they have a variety of surface functional groups which can be tailored to use in different applications [ 16 - 22 ]. In the present study, we attempted to use alizarin yellow-attached magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads as specific adsorbent for aluminium removal from aqueous solutions in a magnetically stabilized fluidized bed. Al(III) adsorption on the alizarin yellow-affinity beads from aqueous solutions containing different amounts of Al(III) ions and at different pHs is reported here. Finally, reuse of the dye-affinity beads is also discussed. Materials and methods Materials 2-hydroxyethyl methacrylate (HEMA), was purchased from Sigma (St. Louis, MO, USA), and was purified by vacuum distillation under a nitrogen atmosphere. The comonomer, ethylene dimethacrylate (EDMA, Merck, Darmstadt, Germany) was used as the crosslinking agent. Magnetite particles (Fe 3 O 4 , diameter < 1 μm) were obtained from Aldrich (USA). Alizarin yellow (3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonic acid, sodium salt mono-hydrate) was purchased from BDH (Poole, UK). All other chemicals were obtained from Merck as analytical grade. All water used in the adsorption experiments was purified using a Barnstead (Dubuque, IA) ROpure LP ® reverse osmosis unit with a high flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANOpure ® organic/colloid removal and ion exchange packed bed system. Preparation of magnetic PHEMA beads Details of the preparation and characterization of the mPHEMA beads were reported elsewhere [ 23 ]. The mPHEMA beads were prepared by suspension polymerization. A typical suspension copolymerization procedure of mPHEMA beads was performed as below: The dispersion medium was prepared by dissolving 200 mg of poly(vinyl alcohol) (PVA; molecular weight: 50.000) within 50 ml of distilled water. The desired amount of 2,2'-azobisisobutyronitrile (AIBN) (0.06 g) was dissolved within the monomer phase 12.0/4.0/8.0 ml (EDMA/HEMA/toluene) with 1.0 g magnetite particles. This solution was then transferred into the dispersion medium placed in a magnetically stirred (at a constant stirring rate of 600 rpm) glass polymerization reactor (100 ml) which was in a thermostatic water bath. The reactor was flushed by bubbling nitrogen and then was sealed. The reactor temperature was kept at 65°C for 4 h. The temperature was then raised to 90°C and kept constant by a thermostated water bath during the polymerization time (2 h). After polymerization, the mPHEMA beads were separated from the polymerization medium. The residuals (e.g., unconverted monomer, initiator and other ingredients) were removed by a cleaning procedure. Briefly, beads were transferred into a reservoir, and washing solutions (i.e., a dilute HCI solution, and a water-ethanol mixture) were recirculated through the system which includes also an activated carbon column, to be assured that the magnetic beads were clean. Purity of the magnetic beads was followed by observing the change of optical densities of the samples (λ: 280 nm) taken from the liquid phase in the recirculation system, and also from the DSC thermograms of the magnetic beads obtained by using a differential scanning microcalorimeter (Mettler, Switzerland). Optical density of uncleaned magnetic beads was 2.63, but after the cleaning operation this value was reduced to zero. In addition, when the thermogram of uncleaned beads was recorded, it had a peak around 60°C. This peak might originate from AIBN, but after application of the cleaning procedure, no peak between 30–100°C was observed on the thermogram. The dry density of the magnetic beads was measured with pycnometer by dispersing the dry beads in ethanol. Alizarin yellow attachment Preparation and characterization of the alizarin yellow-attached mPHEMA beads were reported in our previous paper in detail [ 24 ]. In order to prepare the alizarin yellow-attached magnetic beads following procedure was applied. 5.0 g of dry magnetic beads was weighed and transferred into the SOCl 2 (Carlo Erba, Italy) (10 ml). This reaction medium was boiled in rotary evaporator for 6 h. Then, 2.5 g alizarin yellow was dissolved in absolute ethanol (30 ml). Alizarin yellow-attachment process was performed in ethanol solution for 24 h. At the end of this reaction period, the alizarin yellow-attached beads were removed by filtration and washed with ethanol, water and tetrahydrofuran several times until all the unbound dye molecules were removed. The dye attached beads were stored at 4°C with 0.02% sodium azide to prevent microbial growth. The leakage of the alizarin yellow from the dye-attached beads was investigated within the media at the selected pH in the range of 2.0–7.0. These media were the same which were used in the Al(III) adsorption experiments. The medium with the dye attached magnetic beads was stirred for 24 h at room temperature. Then, magnetic beads were separated from the medium, and the alizarin yellow concentration was measured in the liquid phase by spectrophotometry at 500 nm. Magnetically stabilized fluidized bed procedure Al(III) adsorption studies were carried out in a magnetically stabilized fluidized bed. Beads suspended in pure water were degassed under reduced pressure (by using water suction pump) and magnetically stabilized into a column (10 cm × 0.9 cm inside diameter) equipped with a water jacket for temperature control. The vertically oriented magnetic field was produced by passing DC current through two modified Helmholtz coils (1.5 cm diameter × 2.5 cm thick) spaced 4 cm apart. At a current of 1.6 A (50 W), each coil produced a magnetic field of 40 Gauss. Equilibration of the column was performed by passing four column volumes of phosphate buffer (pH: 7.4) before injection of the Al(III) solution. In a typical adsorption system, 50 ml of the aqueous Al(III) solution was passed through the column containing magnetic beads, by a peristaltic pump for 2 h. After loading, the column was washed with deionized water to wash out Al(III) impurities. The concentrations of the Al(III) ions in the aqueous phases after the desired treatment periods were measured by using a graphite furnace atomic absorption spectrophotometer (AAS 5EA, Carl Zeiss Technology, Zeiss Analytical Systems, Germany). Deuterium background correction was used. Pyrolitic graphite coated tubes were used for AAS measurements. The instrument response was periodically checked with known Al(III) solution standards. The experiments were performed in replicates of three and the samples were analyzed in replicates of three as well. For each set of data present, standard statistical methods were used to determine the mean values and standard deviations. Confidence intervals of 95% were calculated for each set of samples in order to determine the margin of error. In the first group of experiments, the flow rate of the aqueous solution (i.e., 50 ml of the solution with a Al(III) content of 50 mg/L) was changed between 0.5–3.0 mL/min. In the second group of experiments, Al(III) adsorption from aqueous solution was studied at different pH's (2.0–7.0). Adsorption isotherm was also obtained in the magnetically stabilized fluidized bed. Aqueous solutions containing different amount of Al(III) were used in these experiments. The changes in the Al(III) concentration with time was followed to obtain the adsorption curves. The amount of adsorbed Al(III) per dry magnetic beads was calculated by using the concentrations of the Al(III) in the initial solution and in the equilibrium. Desorption and repeated use In all cases adsorbed Al(III) ions were desorbed using 0.1 M HNO 3 solution. In a typical desorption experiment, 50 ml of the desorption agent was recirculated through the magnetically stabilized fluidized bed containing dye-affinity magnetic beads for 1 h. The concentrations of the Al(III) ions in the desorption medium were measured by using a graphite furnace atomic absorption spectrophotometer. The desorption ratio was calculated from the amount of Al(III) adsorbed on the magnetic beads and the final Al(III) concentration in the desorption medium. In order to test the reusability of the dye-affinity magnetic beads, Al(III) adsorption-desorption procedure was repeated ten times by using the same magnetically stabilized fluidized bed. Batch procedure Adsorption of Al(III) from aqueous solution was also investigated in batch experiments. Aqueous Al(III) solution (50 ml) was treated with the magnetic dye-affinity beads at room temperature, in the flasks agitated magnetically at an agitation speed of 600 rpm for 2 h. The suspension was brought to pH 5.0 by adding sodium hydroxide and nitric acid. The pH was maintained in a range of ± 0.1 units until equilibrium was attained. Polymer amount was kept constant at 100 mg per 50 ml. Al(III) determination was performed in water sample in an atomic absorption spectrophotometer coupled to a graphite furnace atomiser. Adsorption values (mg/g) were calculated as the difference in Al(III) ion concentration of the pre- and post adsorption solutions divided by the weight of dry magnetic affinity beads. Results and discussion Characteristics of mPHEMA beads mPHEMA beads (in the size range of 80–120 μm) carrying alizarin yellow were prepared as a specific affinity adsorbent for removal of Al(III) from the water which was used for preparation of dialysis solution. mPHEMA beads used in this study were prepared and characterized in our earlier study [ 24 ]. The main criteria of selection of PHEMA is due to its mechanical strength and chemical stability. With the goal of testing the mechanical stability of the magnetic beads, a sample of these magnetic beads was treated in a ball mill for 60 min. Negligible percentage of the sample was broken. The dry density of the magnetic beads was measured as 1.27 g/cm 3 . The magnetic beads are crosslinked hydrogels. They do not dissolve in aqueous media, but do swell, depending on the degree of cross-linking and on the hydrophilicity of the matrix. The equilibrium swelling ratio (the ratio of the volumes of the microbeads before and after swelling) of the beads used in this study is 34%. The simple incorporation of water weaken the secondary bonds within the hydrogels. This enlarges the distance between the polymer chains and causes the uptake of water. It should be mentioned that the water uptake properties of the mPHEMA beads did not change after Alizarin Yellow attachment. After the attachment of the dye (i.e., alizarin yellow) the size of the swollen beads did not change, but the colour became dark yellow, which is a clear indication of the incorporation of the dye molecules in the structure of the mPHEMA microbeads. As shown in our previous paper, the dye molecules were attached to the mPHEMA beads by covalent bonding via hydroxyl groups [ 24 ]. The mPHEMA beads containing 208 μmol alizarin yellow/g polymer, which was the maximum amount of dye-attachment that we have reached, were used in this study. Alizarin Yellow release from the mPHEMA beads was also monitored continuously. There were no dye release in any of the adsorption and desorption media, which assured that the cleaning procedure used for removal of physically adsorbed alizarin yellow molecules from the mPHEMA beads was satisfactory. Column performance The adsorption capacity at different flow-rates are given in Figure 1 . The adsorption capacity decreased significantly from 17.2 mg/g to 6.9 mg/g polymer with the increase of the flow-rate from 0.5 ml/min to 3.0 ml/min. One of the explanation for such phenomenon would be a faster ligand-metal ion (i.e., alizarin yellow) dissociation rate compared to the association rate. Hence, the adsorbate (i.e., Al(III) ions) would pass through the magnetically stabilized column without adsorption at high flow-rate. Second explanation could be that the increased nonideal flow hydrodynamics of liquid phase and the solid phase for magnetically stabilized fluidized bed. These phenomena can be summarized by the increase of the axial dispersion coefficient in the axial dispersion model [ 25 ]. Figure 1 Effect of flow-rate on Al(III) adsorption. Alizarin yellow loading: 208 μmol/g; Al(III) concentration: 50 mg/L; pH: 5.0; T: 25°C. Adsorption capacity Figure 2 shows the adsorption profile of Al(III) ions. The amount of Al(III) ions adsorbed per unit mass of the polymer (i.e. adsorption capacity) increased first with the initial concentration of Al(III) ions then reached a plateau value at about an initial Al(III) ions concentrations of 50 mg/L, which represents saturation of the active attachment sites (which are available for Al(III) ions) on the beads. The maximum adsorption capacity of Al(III) ions was of 647 μmol/g (17.5 mg/g). Unit mass of the mPHEMA beads carries 208 μmol alizarin yellow which was found by elemental analysis. From the mass-stoichiometry, it seems that one attached alizarin yellow molecule interacts with around three Al(III) ions. Since alizarin yellow has seven coordinating sites of a single sulphur and six oxygen atoms, it can form a ternary complex which is coordinated with water molecules at vacant coordination sites of metal-alizarin yellow complexes. Figure 2 Effects of Al(III) concentration on Al(III) adsorption. Alizarin yellow loading: 208 μmol/g; Flow-rate: 0.5 ml/min; pH: 5.0; Adsorption time: 60 min; T: 25°C. It should be noted that the nonspecific adsorption (adsorption on plain mPHEMA beads) of Al(III) ions was relatively low (0.63 mg/g). mPHEMA beads do not contain ion exchange or chelating groups. Preferred coordination structure and preferred coordinating ligand atom may be utilized for this adsorption. Al(III) ions may interact with Oxygen atoms as the ligand. Diffusion of Al(III) ions into the swollen polymeric structure and retention in the pores may also contribute to this nonspecific Al(III) adsorption. Adsorption isotherms An adsorption isotherm is used to characterize the interactions of each molecule with the adsorbent. In this case it provides a relationship between the concentration of the Al(III) ions in the solution and the amount of Al(III) ions adsorbed on the solid phase when the two phases are at equilibrium. The Langmuir adsorption model assumes that the species are adsorbed at a fixed number of well-defined sites, each of which is capable of holding only one molecule. These sites are also assumed to be energetically equivalent, and distant from each other so that there are no interactions between molecules adsorbed on adjacent sites. Adsorption isotherms were used to evaluate adsorption properties. The Langmuir adsorption isotherm is expressed by Equation 1. The corresponding transformations of the equilibrium data for Al(III) gave rise to a linear plot, indicating that the Langmuir model could be applied in these systems and described by the equation: Q = Q max . b . C eq / (1 + bC eq ) (1) where Q is the adsorbed amount of Al(III) (mg/g), C eq is the equilibrium Al(III) concentration (mg/mL), b is the Langmuir constant (mL/mg) and, Q max is the maximum adsorption capacity (mg/g). This equation can be linearized so that C eq /Q = 1/(Q max . b) + C eq /Q max . (2) The plot of C eq versus C eq /Q was employed to generate the intercept of 1/Q max .b and the slope of 1/Q max . The maximum adsorption capacity (Q max ) data for the adsorption of Al(III) was obtained from the experimental data. The correlation coefficient (R 2 ) was 0.989. The Langmuir adsorption model can be applied in this affinity adsorbent system. Maximum adsorption capacities determined using Langmuir isotherms show that dynamic capacity (25.3 mg/g) was somewhat higher than the batch capacity (12.6 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands. Effect of pH Metal ion adsorption onto specific adsorbents is pH dependent. In the absence of complexing agents, the hydrolysis and precipitation of the metal ions are affected by the concentration and form of soluble metal species. The solubility of metal ions is governed by hydroxide or carbonate concentration. Hydrolysis of metal ions becomes significant at approximately pH 7.5–8.5. Therefore, in the present study, we changed the pH range between 2.0–7.0. The effect of pH on the Al(III) adsorption of this alizarin yellow-attached mPHEMA beads is also shown in Figure 3 . The magnetic mPHEMA beads exhibited a low affinity in acidic condition (pH < 4.0), a somewhat higher affinity between pH 4.0 and 7.0. High adsorption capacities at around neutral pH values imply that Al(III) ions interact with dye molecules not only through the oxygen atoms by chelating, but also electrostatically through sulfonate groups, which are ionized at neutral pH. Figure 3 Effects of pH on Al(III) adsorption. Alizarin yellow loading: 208 μmol/g; Flow-rate: 0.5 ml/min; Al(III) concentration: 50 mg/L; Adsorption time: 60 min; T: 25°C. Competitive adsorption Competitive adsorption of the metal ions from tap water in Ankara and dialysis water (reverse osmosis) was also investigated. The water containing different amounts of each metal ion was treated with dye beads in MSFB. Table 1 and 2 show the adsorbed amounts for each metal ion. The adsorption capacity of the dye-attached mPHEMA beads for Cu(II) and Al(III) ions was higher than that for other ions. But it should also be noted that the extent of adsorption of each type of metal ion is strongly dependent upon their relative concentrations within the medium. Table 1 Aluminium removal from the tap water. Metal Ion % Concentration of Metal Ions (ng/ml) Metal Ion Adsorption (μg/g) Adsorbed Metal Ions (%) Al(III) 80.1 40.3 ± 0.1 98.9 Fe(III) 32.1 10.8 ± 0.2 54.3 Cu(II) 145.3 28.3 ± 0.2 35.1 Cd(II) 0.05 nd - Pb(II) 0.03 nd - Zn(II) 20.4 1.5 ± 0.1 28.6 Adsorption Conditions: Flow-rate: 0.5 ml/min; pH: 5.0; T: 25°C, nd: not determined. Each experiment was repeated three times. Table 2 Aluminium removal from dialysis water. Metal Ion % Concentration of Metal Ions (ng/ml) Metal Ion Adsorption (μg/g) Adsorbed Metal Ions (%) Al(III) 18.96 9.46 ± 0.1 99.7 Fe(III) 0.05 - - Cu(II) 0.82 0.16 ± 0.01 40.0 Zn(II) 1.26 0.44 ± 0.01 69.8 Adsorption Conditions: Flow-rate: 0.5 ml/min; pH: 5.0; T: 25°C. Each experiment was repeated three times. The World Health Organization (WHO) and the European Community (EC) guide values for Al(III) ions for tap water is 200 ng/ml [ 26 , 27 ]. Al(III) concentrations both in tap water and dialysis water are below this value. It should be noted that polymer treatment (i.e, adsorption) significantly decreases the metal content and these purified waters can be used safely especially for the preparation of dialysis solutions. Magnetic dye-affinity beads exhibits the following metal ion affinity sequence: Al(III) > Cu(II) > Fe(III) > Zn(II). Desorption and repeated use Desorption ratios were very high (up to 97.6%) with the eluant system and under conditions used. When HNO 3 is used as a desorption agent, the coordination spheres of chelated Al(III) ions are disrupted and subsequently Al(III) ions are released from the solid surface into the desorption medium. Therefore, we conclude that HNO 3 is a suitable desorption agent for the dye adsorbents, and allows their repeated use. In order to show the reusability of the dye-attached mPHEMA beads, adsorption-desorption cycle was repeated ten times by using the same sample of affinity adsorbent. As can be seen from Figure 4 , adsorption capacities did not noticeable change during the repeated adsorption-desorption cycles. Figure 4 Repeated use of dye-attached mPHEMA beads. Alizarin yellow loading: 208 μmol/g; Flow-rate: 0.5 ml/min; Al(III) concentration: 50 mg/L; Adsorption time: 60 min; pH: 5.0; T: 25°C. Comparison of magnetically stabilized fluidized bed and batch system As can be seen in Figure 5 , maximum Al(III) adsorption from aqueous solution is 11.8 mg/g for batch system and 17.5 mg/g for MSFB system. These results indicated that the adsorption capacity obtained in MSFB system is considerably higher than obtained in batch sytstem. This means, in equilibrium binding experiments, maximum capacity was 38.8% lower as compared to the value obtained in MSFB. This result could be explained in two ways. (i) The dye ligand-Al(III) dissociation rate in the batch system is higher than the association rate in the MSFB system. (ii) Alizarin yellow ligand is found both on the surface and in the pores of the magnetic beads. In the presence of flow, the Al(III) solution is forced from the surface into the pores thus eliminating the surface diffusion. Figure 5 Comparison of MSFB and batch system. Alizarin yellow loading: 208 μmol/g; Flow-rate: 0.5 ml/min; Al(III) concentration: 50 mg/L; Adsorption time: 60 min; pH: 5.0; T: 25°C. Conclusions The medical relevance of aluminium has stimulated the development of cost and time effective separation techniques including polymeric carriers. Magnetic adsorbents have several potential advantages over conventional adsorbents [ 28 - 32 ]. The magnetically stabilized columns require faster processing times and high flow-rates with a much lower operating pressure than a packed bed column. In this study, mPHEMA beads, in the size fraction of 80–120 μm, were produced by a dispersion polymerization of EGDMA and HEMA in the presence of magnetite particles. These novel magnetic beads were then successfully attached with reactive dye-ligand, namely alizarin yellow. The highest dye loading was 208 μmol/g. Al(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was observed at pH 4.0. Al(III) adsorption onto the mPHEMA beads was negligible (0.63 mg/g). Higher adsorption values (up to 17.5 mg/g) were observed using alizarin yellow attached mPHEMA beads for the adsorption of Al(III) ions from aqueous solutions. Al(III) ions could be repeatedly adsorbed and desorbed without significant losses in their adsorption capacities.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC516445.xml
514534	The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events	Background Although lentiviral vectors have been widely used for in vitro and in vivo gene therapy researches, there have been few studies systematically examining various conditions that may affect the determination of the number of viable vector particles in a vector preparation and the use of Multiplicity of Infection (MOI) as a parameter for the prediction of gene transfer events. Methods Lentiviral vectors encoding a marker gene were packaged and supernatants concentrated. The number of viable vector particles was determined by in vitro transduction and fluorescent microscopy and FACs analyses. Various factors that may affect the transduction process, such as vector inoculum volume, target cell number and type, vector decay, variable vector – target cell contact and adsorption periods were studied. MOI between 0–32 was assessed on commonly used cell lines as well as a new cell line. Results We demonstrated that the resulting values of lentiviral vector titre varied with changes of conditions in the transduction process, including inoculum volume of the vector, the type and number of target cells, vector stability and the length of period of the vector adsorption to target cells. Vector inoculum and the number of target cells determine the frequencies of gene transfer event, although not proportionally. Vector exposure time to target cells also influenced transduction results. Varying these parameters resulted in a greater than 50-fold differences in the vector titre from the same vector stock. Commonly used cell lines in vector titration were less sensitive to lentiviral vector-mediated gene transfer than a new cell line, FRL 19. Within 0–32 of MOI used transducing four different cell lines, the higher the MOI applied, the higher the efficiency of gene transfer obtained. Conclusion Several variables in the transduction process affected in in vitro vector titration and resulted in vastly different values from the same vector stock, thus complicating the use of MOI for predicting gene transfer events. Commonly used target cell lines underestimated vector titre. However, within a certain range of MOI, it is possible that, if strictly controlled conditions are observed in the vector titration process, including the use of a sensitive cell line, such as FRL 19 for vector titration, lentivector-mediated gene transfer events could be predicted.	Background Multiplicity of infection (MOI) is a parameter that has been commonly used to predict viral infectivity in a population of target cells. With wild type viruses, an "infectious unit" refers to the smallest amount of virus capable of producing an infection in a susceptible cell. The titre of the original suspension is defined as the number of infectious units per unit volume of the preparation [ 1 ]. In the field of gene therapy where viral vectors are used for gene transfer, MOI was adopted to represent the ratio of input infectious units (titrated on the target cell line) to the number of cells available for transduction [ 2 ]. Ideally, there should be a simple linear relationship between the viral vector titre, its dilution, the volume of viral vector suspension used, and the proportion of cells infected, taking into account the probabilistic nature of the infective process when the number of viral vector particle approximates the number of cells. However, at present, the number of viable vector particles (or vector titre) in a given vector stock is determined by a vector-mediated transduction process, which is of a non-linear nature and can be influenced by various factors. If MOI is based on vector titre that is "variable", then MOI is complicated by all of the factors that influence vector titration and determination. Unfortunately, the extent of which is poorly understood. Recently, lentivirus-based gene transfer vectors have been developed and have shown considerable promise for gene therapy research. It is evident that this vector system has several distinct advantages, and rapidly emerges as the vector of choice for in vitro and in vivo gene therapy studies [ 3 , 4 ]. Most current lentiviral vectors in use are based on Human Immunodeficiency Virus (HIV) type 1. A transient, three or four-component, HIV-1 based vector system consisting of one or two packaging constructs, a transfer vector and a Vesicular Stomatitis Virus G glycoprotein (VSV-G) envelope has recently been described and widely used [ 5 - 10 ]. Several reports have demonstrated that the HIV-based vectors effectively transduced dividing and non-dividing cells in vitro and in vivo including hematopoietic stem cells [ 7 , 11 ], terminally differentiated cells such as neurons [ 9 ], retinal photoreceptors [ 8 ], muscle, liver cells [ 5 ] and dendritic cells [ 12 ]. Other lentivectors, such as those based on the feline immunodeficiency virus (FIV) [ 13 ], equine infectious anaemia virus (EIAV) [ 14 ], caprine arthritis/encephalitis virus (CAEV) [ 15 ], Jembrana disease virus (JDV) [ 7 ], bovine immunodeficiency virus [ 16 ] and visna virus [ 17 ], are examples of recently developed non-primate lentiviral vectors that have also demonstrated efficient gene transfer to various types of cells. Just as with Moloney murine leukaemia virus (MoMLV) based retroviral vectors, many variables could theoretically affect the measurement of infectivity of lentiviral vector particles, such as target cell type, number, cycle, other modulators of cell membrane ingredients, the time needed for vector uptake and vector viability/susceptibility, half life during the transduction process or even Brownian motion in which the vector makes way to the target cell [ 18 ]. In addition, the issue of particle variation within the population of artificially assembled vector "infectious" units could be a contributory factor to between-preparation variation in the predictability of their infectious behaviour. Arai et al (1999) found that the ratio of cells transduced with the VSV-G-pseudotyped retroviral vectors based on MoMLV correlated with the result predicted from a Poisson distribution [ 9 ]. Generally with retroviral vectors using an ecotrophic or amphotrophic envelope, MOI at 1–3 is commonly used and results in around 30% of cells being transduced. The efficiency of gene transfer reaches a plateau after this. Higher MOI may reduce the number of transduced cells [ 3 , 19 ]. However, with lentiviral vector-mediated gene transfer, experiments employing MOI even greater than 1000 have been explored [ 12 ]. The rational behind the usage has obviously distinguished lentiviral vector from MoMLV based retroviral vectors. Unfortunately, there are, at present, no data available as to how lentiviral vectors behave in an in vitro transduction process, and how the variables affect vector titre determination and MOI usage. In this study, we characterised factors that influenced the in vitro vector titration process, including the number of target cells being transduced, total number of viral vector particles, inoculum volumes (well beyond the depth of relevance to diffusion), vector decay and the period of vector adsorption (and thus vector decay). We also examined the use of various MOIs on several commonly used cell lines and tried to establish the relationship of MOI with the efficiency of gene transfer. Methods Cell cultures Cell lines used in this study were a fetal rat liver carcinoma cell line, FRL 19; a human embryonic kidney cell line, 293 and its derivative, 293T; and a murine embryonic fibroblast cell line, NIH 3T3. FRL 19 was maintained at 37°C in Ham and Dulbecco's modified Eagle's medium (1:1 ratio, DMEM; Life Technologies Inc) containing 2 mM glutamine, 4% Fetal Calf Serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin, 1 μg of fungizone per ml (Ham and DMEM), 10 -7 M of insulin, and 10 -7 M of dexamethasone in a 5% CO 2 incubator. All other cells were maintained at 37°C in DMEM containing 2 mM glutamine, 10% Fetal Calf Serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin, similarly in a 5% CO 2 incubator. 293, 293T and NIH3T3 were maintained in DMEM containing 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomcycin at 37°C similarly in a 5% CO 2 incubator. Cells were seeded at 5 × 10 5 on 10 cm or 7.5 × 10 5 on 15 cm plate and were at 70 – 80% confluence at the time of transfection or transduction. Viral vector production Replication-defective retroviral particles were generated by transient co-transfection of 293T cells with the three plasmids (pHR' CMVGFP or pHIV-CSGFP, pCMVΔR8.2 pr pCMVΔR8.9 and pHCMV-G), using a CaPO 4 precipitation method as we previously reported [ 21 ]. Briefly, 293T cells were grown on 10 cm plates to 70–80% confluence and co-transfected with 10 μg pHCMV-G, 10 μg pHR' CMVGFP or pHIV-CSGFP and 20 μg pCMVΔR8.2 or pCMVΔR8.9. The plasmid DNA was diluted into 250 mM CaCl 2 in 1/10-TE buffer (1 mM Tris HCl, 0.1 mM EDTA, pH 7.6) in 0.5 ml before an equal volume of 2× HBS (140 mM NaCl, 1.5 mM Na 2 HPO 4 , 50 mM HEPES, pH 7.05) was added and mixed by gently bubbling air through the mixture for 1 min. The solution was then added drop-wise onto the cells (100 μl per 1 ml of culture media). The cell cultures were rinsed with PBS and given fresh media within 10–12 hr after initiating transfection. The medium was harvested 48 hr post-transfection, centrifuged at low speed to remove cell debris and filtered through a 0.45 μm filter. The supernatant was stored at 4°C no more than 24 hr before it was used for transduction. Ultracentrifugation This was performed as reported previously [ 20 , 21 ]. Briefly, 30 mL of vector-producing cell (VPC) supernatant was added to each polypropylene ultra-centrifugation tube (6 × 30 mL), and ultracentrifuged at 50,000 g for 2 hr at 4°C on AH629 rotors in a Beckman refrigerated centrifuge. After centrifugation, the tubes were promptly removed and supernatant decanted. The viral pellet was resuspended in 0.6 mL of DMEM and stored at -20°C. In vitro transduction and determination of lentivector titre This was performed as we previously reported [ 20 ]. Briefly, cultured 293T cells were seeded at 5 × 10 5 cells and transduced with serially diluted and concentrated viral vector stocks 16–18 hours after seeding when cells were about 70% confluent. For each transduction, 8 μg/mL of polybrene (Sigma) was included in the transducing inoculum. Forty-eight hours after transduction, EGFP positive fluorescent cells were counted using epifluorescent microscope (Nikon eclipse E600, Japan) with the fluorescein isothiocyanate (FITC) excitation-emission filter set at 470 nm. The viral vector titre was determined as the average number of EGFP positive cells per 20 1-mm 2 fields multiplied by a factor to account for dilution of the viral stock as well as plate size and thus total cell number. Alternatively, 48 hours after transduction, cells were harvested, resuspended and sent for FACs analyse at a local FACS facility (Queensland Institute of Medical Research, QIMR, Brisbane, Australia). Transduction – studies of target cell volume and number 293T cells at 1 × 10 3 , 3 × 10 4 or 1 × 10 5 per well were seeded in triplicate in 24-well plates. Transduction was performed with the same stock of viral supernatant using volumes of 100 μl, 300 μl and 1 ml for 2 hours in the presence of 10 μg/ml polybrene. After the incubation period the cells were washed with fresh growth medium twice and allowed to grow for 2 days before the cells were trypsinised and fixed with 2% formaldehyde + 0.2% glutaraldehyde in PBS. EGFP positive and total cell numbers were counted with a haemocytometer using epi-fluorescence microscopy. Transduction – studies of variable vector-cell contact and adsorption periods 293T cells were grown in 24-well plates to approximately 70% confluence. The cells were incubated with 500 μl of pHR' CMVLacZ supernatant for 10 min, 30 min, 1 hr, 2 hr, 4 hr and 17 hours. After the indicated incubation period the viral supernatant was removed and replaced with fresh media. Forty-eight hours post-transduction, cells were stained to check for the presence of LacZ with the following solution: 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 .3H 2 O, 2 mM MgSO 4 , and 1 mg/ml X-gal in PBS. Blue cells or colonies were counted as positive for gene transfer. Transduction – studies of vector decay Cell-free viral vector-containing supernatant was incubated at 37°C for 30 min, 2 hr, 4 hr, or 24 hr prior to being used as the transducing medium (500 μl), with experimental samples in triplicate. 293T cell at 70% confluent cultures were exposed to the transducing media for 2 hours, after which the inoculum was removed and the cultures replenished with fresh media. Forty-eight hr post-transduction, cells were stained to check for the presence of LacZ with the X-gal solution. Blue cells/colonies were counted in 3 fields and the average used as the titre at that time point. Transduction – studies of MOI and transgene expression 293T cells were plated in a 10 cm plate at 1 × 10 5 cells/plate. Transduction was performed with viral vector stocks at a MOI of 2, 4, 8, 16 and 32 in the presence of 10 μg/ml polybrene (Sigma). Transduced cells were passaged every three days and EGFP positive cells sorted at a local FACS facility (QIMR, Brisbane, Australia). Flow cytometry Flow cytometry analysis was performed to evaluate the expression of lentivirus vector-mediated gene transfer. Cells were washed with PBS, and then fixed with 1% paraformaldehyde before the analysis. Samples were analysed on a FACScan flow cytometry in QIMR. Results The inoculum volume of the vector and the number of target cells affect vector titre determination, but not proportionally Figure 1 shows that during the lentiviral vector titration process, the higher the inoculum volume of the vector (ie. more viral vector particles) the more numbers of positively transduced cells. This was true over a range of target cells tested from 1 × 10 3 to 1 × 10 5 cells/ml. The results suggest the higher inoculum volume of the vector the more opportunity for a viral vector to reach a given target cell. Figure 1 Higher inoculum volumes (more vector particles) and increased number of target cells resulted in higher efficiency of gene transfer. This was true over a range of target cells from 1 × 10 3 to 1 × 10 5 and volumes from 0.1 ml to 1 ml. However the increase in gene transfer was not proportional to the increase in inoculum volume. e.g. a 10 fold increase in volume resulted in only a 3.7 to 4.7 fold increase in transduction efficiency. The values represent mean ± SD (n = 4). However, the results contradicted the data from an amphotropic MoMLV viral vector-mediated gene transfer where it was found that by keeping the virus vector concentration constant while the inoculum volume varied, the infectivity remained the same [ 19 ]. This discrepancy was not accounted for by the depth of fluid as in the present experiments, in the wells (area = 2 cm 2 ) of the cell culture, the depth of the fluid varied from 0.5 mm (with a volume of 0.1 ml) to 1.5 mm for the 0.3 ml volume, and a depth of 5.0 mm for 1 ml of the vector preparation. All of these depths were well beyond the diffusion limit of relevance to the adsorption of 95% of a retrovirus preparation. This was because the rate decreased with the square of the depth, equating to 0.16 mm for a 2.5 hours adsorption period [ 2 ]. Similarly, vector titre was also affected by the number of target cells used in the vector titration process. A very significant increase in vector titre was noticed with increasing the cell numbers, but the increase was also not proportional. For a 30-fold increase in target cell number between 1 × 10 3 and 3 × 10 4 there was only an average of 9.17-fold increase in total number of transduced cells (for all transducing volumes). For a further 3.3-fold increase in cell number exposed in the same area, there was only a further 2.3 fold increase in total number of transduced cells. Thus, overall for a 100-fold increase in cell numbers (from 1 × 10 3 – 1 × 10 5 ) exposed to vectors there was only a 21.3-fold increase in total number of transduced cells. Interestingly, the increase of the number of positively transduced cells was not proportional to the increase of the vector inoculum volume. The increase in the number of transduced cells was proportionally less than the increase in inoculum volume, e.g. a 10-fold increase in inoculum volume resulted in only a 3.7 to 4.7-fold increase in the number of positively transduced cells. Vector decay and the period of vector adsorption to target cells were significant factors in influencing the transduction process The length of period of vector adsorption to target cells was shown to alter the transduction efficiency significantly. As the incubation period increased so did the number of transduced cells (Figure 2a ). At 4 hours less than half of the active vectors had adsorbed on to the cells. Since vector adsorption to cells was often protracted, the issue of thermostability of the vector preparation arose as a negative modulator of transduction efficiency with increasing time, thereby producing further variation in the estimated titre and thus the "MOI". Figure 2 The period of adsorption (a) and vector decay (b) were significant factors in determining transduction efficiency. The duration of the adsorption period was also shown to alter the transduction efficiency significantly. As the incubation period increased so did the number of transduced cells. At 4 h less than half of the active vectors had adsorbed to the cells. To estimate the t (1/2) of the vector system used here, we pre-incubated the inoculum for increasing periods of time before applying aliquots to the target cell monolayer. By applying the following equations V A = V A o exp (-k d t) and t (1/2) = ln(2)/k d to the data, {where V A is the concentration of active virus at time t, V A O is the initial concentration of active virus, and K d is the virus decay rate constant}, the half-life of the vector was in the 8–9 hr range. The values represent mean ± SD (n = 4). To estimate the half time (t (1/2) ) of the vector system used here, we pre-incubated the inoculum for increasing periods of time before applying aliquots to the target cell monolayer for vector titre determination. The length of time for which the viral supernatant harvest was left at 37°C (in a cell-free environment) prior to use, noticeably affected the value of the vector titre (Figure 2b ). The viral vector activity decayed logarithmically with time. By applying the following equations: V A = V A o exp (-k d t) and t (1/2) = ln(2)/k d to the data, {where V A is the concentration of active virus at time t, V A O is the initial concentration of active virus, and K d is the virus decay rate constant}, the half-life of the vector was in the 8–9 hours range. This is the first time that lentivector stability has been examined. This estimation was twice as long as that for wild-type HIV [ 1 ], suggesting that lentivector is much more stable. Variations in viral vector titration further complicated the use of MOI for predicting gene transfer events Lentiviral vector titre (transducing unit per millitre, TU/ml) was calculated using the number of TU/ml times the dilution factor of the vector stock, divided by the volume of vector used in the transduction. As shown in the above results, the number of positively transduced cells changed when the transduction conditions varied. Therefore, the vector titre was affected by inoculum volume, vector stability and target cell numbers. If vector titres were to be calculated using the existing formula that was developed based on retroviral vector-mediated gene transfer, i.e. EGFP-positive cells (TU) ÷ volume of vector inoculum (ml), the titre of the original vector suspension would result in absurdly different figures (see Table 1 ), with ranges from 2.2 × 10 2 TU/mL to 1.2 × 10 4 TU/mL for the same viral suspension, more than a 50 fold difference. Likewise, because MOI is based on vector titre (MOI = titre × TD volume / number of cells), the use of MOI was thus affected. Table 1 Different titres and MOI were obtained for the same vector stock when different numbers of target cells and volumes of inoculum were used. The number of positively transduced cells and thus the transduction efficiency, was also affected by the number of target cells in the transduction process, eg.: a thirty-fold increase in cell numbers resulted in a 53% decrease in efficiency. The transduction efficiency was highest with the smallest cell number and largest inoculum volume. Titre TU/mL (followed by MOI) Number of target cells 1 mL of VI Vol. 0.3 mL of VI Vol. 0.1 mL of VI Vol. 2.24 × 10 2 ( 0.224 ) 3.96 × 10 2 ( 0.119 ) 6.08 × 10 2 ( 0.061 ) 1 × 10 3 2.14 × 10 3 ( 0.071 ) 3.77 × 10 3 ( 0.038 ) 5.14 × 10 3 (0.017 ) 3 × 10 4 5.58 × 10 3 ( 0.056 ) 7.79 × 10 3 ( 0.023 ) 1.19 × 10 4 ( 0.012 ) 1 × 10 5 TU – Transducing Unit; VI Vol – Volume of Inoculum. Considerable differences existed in the sensitivity of lentiviral vector-mediated gene transfer in several conventional cell lines The sensitivity of lentivector-mediated EGFP gene transfer to commonly used target cell lines has never been directly compared previously. In this study, 3 commonly used cell lines plus a new cell line FRL-19 were included for comparison. All cells were seeded in 12 well plate at 5 × 10 4 cells/well 16–18 hours before transduction. Concentrated viral vectors with unknown titre were added to each well at 50 μl, 100 μl, 200 μl, and 400 μl. Medium was changed every day. All cells were harvested 72 hours after transduction, washed twice with PBS, and then analysed by FACS. Figure 3 showed that the percentage of EGFP positive cells was 88.1% for FRL-19 cells, 52.9% for 293T cells, 34.7% for NIH 3T3 cells, and 27.8% for 293 cells respectively when 50 μl viral vector was used for transduction. Clearly transduction efficiency of lentivector-mediated EGFP gene transfer to FRL-19 was the highest amongst the four cell lines tested. It reached 96.7% when 400 μl of viral vector was used while the transduction efficiency of lentivectors was only 87.9% for 293T cells, 77.1% for NIH 3T3 cells, and 63.9% for 293 cells for the same volume of vector (Fig. 3A ). When a third generation of lentiviral vector packaging system (pMDg/p, pRSV-Rev, gifts from Professor Didier Trono, Department of Genetics and Microbiology, CMU., Switzerland) were used to package a HIVCS-CMV-EGFP vector, a very similar transduction efficiency was obtained (Zhang et al., unpublished data). These results convincingly demonstrated that conventional cell lines were less sensitive to lentiviral vector-mediated gene transfer than FRL19, thus grossly underestimating vector titre. Figure 3 Efficiency of lentivector-mediated gene transfer to commonly used target cell lines (A) under different MOI (B). Four cell lines were seeded at 5 × 10 4 /well in 12 well plates. Several different inoculum volumes of lentivectors without known titre (A) or with known titre, ie.: different MOI (B) were added were added to each well (A) or as indicated. The media was changed daily. Cells were harvested three days after transduction, and washed three times with PBS. Transduction efficiency of lentivectors in different cell lines was obtained using flow cytometric analysis. Data represents mean value ± SD (n = 4). The sensitivity of cell lines to lentivectors was generally MOI dependent We further examined whether the sensitivity of these cell lines to lentivectors-mediated EGFP gene transfer was dependent on the MOI. All four cell lines were seeded in 12 well plate at 5 × 10 4 cells/well 16–18 hrs before transduction. Viral vectors with known titre were added to each well at different MOI (MOI = viral titre/cell number). Medium was changed every day, with cells harvested 72 hrs after transduction, washed twice with PBS, and then examined by FACS analysis. Figure 4a shows that transduction efficiency of lentivectors was higher on the FRL-19 cell line than the other three cell lines. Transduction efficiency was 67.4% in FRL-19 cells, 33.1% in 293T cells, 23.1% in NIH 3T3 cells, and 8.7% in 293 cells at a MOI of 32. Generally, it was the higher the MOI, the higher the transduction efficiency (Fig 3B ). Discussion We showed in this study that a number of factors within the vector titration process, ie.: the volume of inoculum, the number of target cells, cell type and viability/susceptibility, vector exposure time for uptake and vector half life affected vector titre determination. We were also surprised to find that the volume of inoculum (with a constant virus concentration) played such an important role in the determination of transduction efficiency. It has been demonstrated that above the cell's surface in MoMLV based retroviral vector mediated gene transfer, a fluid layer of 0.1–1 mm thick remained stationary, and this layer is seen to be the major source of origin of the transducing elements. The large effects seen with non-agitated cultures in the present series of experiments with lentiviral vectors indicated some fundamental differences in the processes of the transduction pathways of MoMLV based retroviral vectors and lentivirally derived vectors. During the transduction process, the rate of collision between the virions and the surface of the target cells could be predicted from Brownian theory even when the viral suspension was being shaken continuously [ 22 ]. This appears to suggest that successful transduction depends on the concentration of virus and not the overall number of virions present, due to the layer effect. The fact that viral vector titre may vary from the transduction process and that the MOI was calculated based on the viral titre, suggested that different vector titres and MOIs could be generated from a single lentivector stock, making direct comparison of data difficult, especially when the difference in vector titre was as high as 50 fold. Therefore, the titre obtained this way obviously did not represent the true value of active vector concentration. Rather, it was grossly underestimated when commonly used cell lines were used as target cells for vector titration. The viral stocks of most lentiviral vectors are generally produced from a 293 or 293T cell lines and the titre calculated by determining the number of foci (effect of the marker gene expression) produced in the cell line [ 23 ]. For example, if 100 μl of the vector suspension gives rise to 1 × 10 5 cells positive for a given marker gene expression, then the titre of the vector stock would be 1 × 10 6 TU/ml. When this vector stock is further used to transduce a new cell line, MOI is then determined by simply dividing the number of viral vector units added (ml added × TU/ml) by the number of target cells added (ml added × cells/ml). The average number of viral vector particles per cell in a transduction experiment could be less than 0.1 or more than 1000 depending upon how the experiment is designed. However, recent research showed that if MOI is too low, one may not get enough gene transfer and transgene expression [ 24 ]. If MOI is too high, the efficiency of gene transfer may not be very high, but many copies of transgene may integrate into the chromosomes of the target cells instead, thus causing chromosomal instability [ 24 ]. Employing MOI from 0–32, we demonstrated that efficient transduction of four different cell lines (293, 293T, NIH3T3, FRL19) resulted in a near liner relationship of MOI to transduction efficiency, the higher the MOI, the higher the transduction efficiency. This was somewhat surprising and contradicted traditional MoMLV based vectors, which showed an obvious plateau when the MOI was increased to about 3 [ 3 ]. The reason for this is unclear, but the fact that lentiviruses are more complicated retroviruses, having more sophisticated machinery for replication and integration than MoMLV, as well as that lentiviral vectors were exploiting the pseudotyped envelope (VSV-G utilises a different receptor), may probably explain the difference in gene transfer efficiency. The VSV-G envelope, binds to its target in cell membranes which are known to be phospholipids, such as phosphatidylcholine (PC) and phosphatidylserine (PS), (the receptors for VSV-G). PC is the most abundant membrane phospholipid while PS domains are present in much smaller quantity but bind more strongly and fuse faster with the VSV-G protein [ 25 ]. This issue is probably one of the most overlooked variables in vector transduction. Membrane phospholipid movement is highly dynamic. Its biosynthesis and degradation are very much dependent on cell type and positions in the cell cycle and/or metabolic activity. Also, the rate of degradation is rapid in G 1 , slows drastically during S phase, and picks up the pace again as cells exit mitosis and re-enters G 1 [ 26 ], which suggests that the cell cycle phase may be an important variable for VSV-G protein coated lentiviral transduction, and may contribute to the time dependence of the transduction efficiency observed in the present experiments. A further contribution to the volume effect may be increased cellular phospholipid uptake from the serum in the expanded volume of medium used for the delivery of the increased total vector or possibly enhanced phospholipid synthesis in a more generous nutritional environment. Cells double their phospholipid mass while maintaining the correct relative composition prior to cytokinesis [ 27 ]. Theoretically, during the intermitotic period the target cells will double the number of target binding sites for the viral vectors as well as allowing a period with the more favourable conditions (DNA synthesis) for integration. The amount of PC in the total membrane mass varies from 40–80% of the total P-lipid, depending on the cell type [ 27 ] and this variation may explain the discrepancies in transduction efficiencies observed with different cell lines using inocula of the same volume and titre of vector. In the real world of gene transfer experiments, transduction conditions will be optimised to achieve the maximum efficiency. Generally, a high MOI is needed for satisfactory levels of gene transfer. Ideally, with a MOI of 2, every single cell might be expected to experience an average of two gene transfer events in a given transduction experiment, but probabilistic considerations of viral and vector-cell interactions ensure that this does not occur (i.e. only 67% of the cells would be "infected"). As seen in the current data, however, the efficiencies of transduction are very much less than the theoretical outcomes. Our study with lentiviral vector convincingly showed that the higher the MOI, the higher the efficiency of gene transfer and the level of gene expression. However, experiments employing MOI even greater than 1000 have still resulted in less than 100% of cells transduced [ 11 , 28 , 29 ] indicating the presence of unexplained variables in the cell dependence of the transduction process. Conclusions MOI is only a useful term for predicting transduction efficiency under very carefully defined experimental conditions. The assumption is not valid that changes in any one of the variables shown to be important in the in vitro vector titration process will cause proportional changes in the magnitude of the transduction efficiency. It is thus evident that MOI is not applicable as a simply manipulable quantity in most gene therapy uses of the lentiviral vector system. Since clinical applications are an important outcome of gene transfer manipulations, and ultimately this may be done by in vivo delivery, the awesome task of evaluating the efficiency of transduction via this route will require considerable ingenuity. If MOI for lentiviral vector transduction has to be used for rigorous comparisons of data, then the specific experimental conditions for vector titration, with using the most sensitive cell lines, such as FRL 19, must be strictly observed for infectivity outcomes to be predictable. List of Abbreviations CAEV, caprine arthritis/encephalitis virus; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; EIAV, equine infectious anaemia virus; FCS, Fetal Calf Serum; FITC, fluorescein isothiocyanate ; FIV, the feline immunodeficiency virus; HIV, Human Immunodeficiency Virus; JDV, Jembrana disease virus; MOI, Multiplicity of Infection; MoMLV, Moloney murine leukaemia virus; PC, phosphatidylcholine; PS, phosphatidylserine; VPC, vector-producing cell; VSV-G, Vesicular Stomatitis Virus G glycoprotein; Competing interests None declared. Authors' contributions BZ performed the use of MOI to predict gene transfer events in the four cell lines; PM performed titration of lentiviral vectors; HJ performed the statistics and Table 1 . KE helped with design of the experiments in examining various conditions in in vitro transduction; GC provided some in BZ and HJ's work; M West provided advice on analysis of the data and manuscript writing; M Wei helped with the design and day to day supervision of all the experiments, assisted with analysing the data and prepared and prove read the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514534.xml
548505	Influence of intense multidisciplinary follow-up and orlistat on weight reduction in a primary care setting	Background Obesity is the most common health problem in developed countries. Recently, several physicians' organizations have issued recommendations for treating obesity to family physicians, including instructions in nutrition, physical activity and medications. The aim of this study was to examine if effective weight-reducing treatment can be given by a family physician. It compares regular treatment with intensive treatment that include close follow-up and orlistat treatment. Methods The study was conducted in three primary care clinics. 225 patients were divided into three groups according to their choice. Group A received a personal diet with fortnightly meetings with the family physician and dietitian and orlistat treatment. Group B received a general diet, monthly meetings with the family physician only and orlistat treatment. Group C received a personal diet, monthly meetings with the dietitian only and no drug treatment. The primary endpoint was reduction of at least 5% of the initial weight during the study period. Results A greater percentage of patients in group A achieved their weight reduction goals than in other groups (51%, 13% and 9% in groups A, B and C, respectively, p < 0.001). There was a significant reduction in triglycerides in all groups, a significant reduction of low density lipids (LDL) in groups A and B and no significant difference in high density lipids (HDL) in any group. Conclusions Significant weight reduction was obtained in a family physician setting. Further research is needed to evaluate if, by providing the family physician with the proper tools, similar success can be achieved in more clinics.	Background Obesity is the most common health problem in developed countries [ 1 ]. It is a chronic disease and should be treated as such. Its prevalence is increasing worldwide [ 2 ]. In the United States, it is estimated that 64% of the adult population is either overweight or obese with a body mass index (BMI; kg/m 2 ) above 25 [ 3 ]. The rate of obesity is increasing [ 4 ] and has risen by more than 75% in the USA since 1980 [ 5 ]. In 2001, the prevalence of obesity (BMI ≥ 30) was 20.9% vs 19.8% in 2000, an increase of 5.6% [ 6 ]. In Israel, according to a survey of the Nutrition Department of the Ministry of Health, 55% of adult (ages 25–64) women and 59% of adult men have a BMI above 24.9 [ 7 ]. Obesity is associated with increased prevalence of many serious chronic diseases such as diabetes mellitus, hypertension, dyslipidemia, and coronary heart disease [ 8 , 9 ]. It may be responsible for approximately 300,000 deaths in the USA per year [ 10 ]. In the Nurses Health Study, the 14-year mortality rate for women with a BMI greater than 32 was more than double that of women with a BMI of less than 19 [ 11 ]. Obesity now ranks second only to smoking as a cause of preventable death but, soon, obesity may surpass smoking as the leading cause of preventable death in the USA [ 12 ]. In the USA, 19% of deaths from coronary disease and 62% of deaths from diabetes can be attributed to obesity [ 13 ]. The risk of death from all causes increases in moderately and severely overweight men and women of all age groups [ 14 ]. Diet and exercise have limited effectiveness on long-term maintenance of weight loss [ 15 ]. Within five to seven years, 95% of all patients regain the lost weight or more [ 16 ]. Pharmacotherapy in combination with a reduced energy diet improves long-term efficacy [ 17 ]. Loss of 5–10% of their initial body weight substantially improves the health of obese patients and modifies their cardiovascular risk factors [ 8 , 18 ]. Despite growing information on the pathophysiology of obesity and its high prevalence, obesity and obesity-related diseases are still under-diagnosed and untreated by family physicians [ 19 ]. Most family physicians cite lack of time, resources, reimbursement from insurance companies, or knowledge of effective interventions as significant barriers [ 20 ]. The intervention of primary physicians during a ten minute physician/patient encounter and telephone consultation with a community dietitian resulted in a significant decrease in the weight of patients [ 20 ]. Recently, several physicians' organizations have issued recommendations for treating obesity to family physicians, including instructions in nutrition, physical activity and medications. Such recommendations were based on a number of studies that proved the effectiveness of family physician weight-reduction programs, when based on the readiness of patients to make necessary lifestyle changes and use of appropriate techniques to increase the willingness of the patient to make necessary changes [ 21 - 24 ]. The purpose of this study was to examine if more efficient and effective weight-reducing treatment can be given in the family doctor setting. The study compare a non-pharmacological intervention with drug intervention (orlistat) and compare regular management with more intensive family physician based management.. Methods Study design The study was conducted in three primary care clinics in an urban area in central Israel. The family physicians who took part in this study participated in 80 hours CME course dealing with obesity treatment in Israel. The patients were divided into three groups according to their choice. Patients in groups A and B were treated with orlistat at 120 mg TID. Orlistat (Xenical ® ) is a lipase inhibitor for obesity management that acts by inhibiting the absorption of dietary fats. The patients in group A received in addition a personal reduced-energy diet and met with a family practitioner and a clinical dietitian once every two weeks. The personal diet was created according to the daily schedule and preferred foods of the individual, emphasizing low-fat foods. The patients received instructions regarding the importance of physical activity and, at each meeting, realistic intermediate goals for achieving two or three small changes in eating habits and physical activity, based on the patient's desires, were determined. The obstacles to change and ways to overcome them were discussed. Support, based on improvements in the patient's health parameters such as an improvement in a blood test, was given. Some of the patients helped with self-criticism, by keeping food and physical activity diaries and by grading their own goal accomplishments. Patients were taught how to resist temptation and reward themselves for success. We also recruited the support and encouragement of the patient's family. At each meeting, the time of the next meeting was determined and it was emphasized that the most important thing for the patients to do was to attend the meetings, even if their goals were not achieved. Group B patients were treated with 120 mg orlistat t.i.d., a general formulated reduced-energy diet and follow-up by the family physician once every four weeks for weighing and prescription renewal. In groups A and B, patients were asked at each meeting if any side effects of orlistat appeared. Patients in group C were given a personal low-calorie diet, designed according to their preferences, and followed-up by a clinical dietitian once a month. The prescribed daily caloric intake was equal in the three groups and was 1200 calories per day for women and 1500 calories per day for men. Before the intervention, every patient received an explanation of the three treatments, the importance of reducing weight, and how excess weight affects their health. All were instructed about the recommended rate of weight loss and a final weight reduction goal of at least 5% of their initial weight within half a year was established. Patients who achieved the 5% reduction goal before the end of the study could choose either to stop the intervention or to complete the study period. Patients Obese (BMI > 30) patients of either sex or patients with a BMI above 27 plus two or more cardiovascular risk factors, aged 20–75 years, were eligible for the study. Patients were excluded if they were pregnant or lactating or if they had any contraindication against using orlistat (chronic malabsorption syndrome, cholestasis, pancreatic disease). Before the intervention, patients underwent an initial screening that included recording of a complete medical history, measurement of vital signs, body weight and height, and calculation of the BMI. Laboratory analysis included a lipid profile. The readiness of the patient to receive treatment was assessed. At the beginning of the intervention, all participants were in the third stage of readiness ("the preparation stage") according to the Transtheoretical Model of Behavior Change. An adverse event of any dose of orlistat was considered serious if it resulted in death, was life-threatening, required hospitalization, or resulted in significant disability. Efficacy measures The main measure was weight loss. Each patient was weighed during each meeting. The primary endpoint was reduction of at least 5% of the initial weight during the study period (six months). Achievement of this goal was defined as successful treatment. Another measure was improvement in the lipid profile. The second lipid profile was done to half of the patients, only those who had dyslipidemia in the first profile had been offered a second profile. Statistical analysis Data on patients' background and weight-reduction results between groups were compared using the Chi square test. Continuous data of the groups, i.e., measurements at the beginning of the study and continuous background data, were compared using one-way tests (ANOVA). When significant statistical differences were found among the three groups, the Tukey Post Hoc test was used to examine the statistical difference between each group of two. ANCOVA was also used and, if there was a difference amongst the groups, the significance (for the measured parameter) was checked using the Bonferonni technique. Results Two hundred and twenty-five patients participated in the study. Their demographic characteristics, history of cardiovascular disease, and initial lipid profile are shown in Table 1 . There were no significant differences between groups in average age, initial BMI or gender of the participants. The average length of follow-up and the number of meetings varied among groups. There were no cases of significant side effects that required stopping orlistat treatment of any of the participants. Table 1 Participants' demographic data, initial lipids profile, by treatment group Group A Group B Group C P* value Number of patients 62 112 51 Age (years +/- SD) 47.3 ± 11 46.8 ± 12 51 ± 9.6 NS Gender (% female) 71 74 61 NS Ischemic heart disease (%) 0 4 0 NS Hypertension (%) 44 51 27 P < 0.05 Diabetes mellitus (%) 9 18 20 NS Dyslipidemia (%) 16 38 66 P < 0.001 Initial body weight Initial body mass index (BMI; kg/m 2 +/- SD) 33 ± 3.8 34 ± 4.4 31 ± 3.6 P < 0.01 34 > 31(B > C) Initial triglycerides (mg/dl, +/- SD) 170 ± 53 184 ± 49 255 ± 205 P < 0.01 170 <255> 183 (A < B > C) Initial low density lipoproteins (LDL; mg/dl, +/- SD) 150 ± 30 156 ± 36 152 ± 44 NS Initial high density lipoproteins (HDL; mg/dl, +/- SD) 42 ± 7.0 44 ± 6.7 47 ± 14.9 NS Average length of treatment (weeks, +/- SD) 13 ± 12.0 9 ± 4.7 23 ± 12 P < 0.001 23 > 13 > 9 (C > A > B) Number of meetings with physician/dietitian (+/- SD) 4.3 ± 2.0 3.5 ± 1.5 5.2 ± 2.9 P < 0.001 5.2 > 3.5 (C > B) NS = Not significant. Group A – Orlistat, a personal reduced-energy diet and a meeting with a family practitioner and a clinical dietitian once every two weeks. Group B – Orlistat, a general formulated reduced-energy diet and follow-up by the family physician once every four weeks. Group C – a personal low-calorie diet and follow-up by a clinical dietitian once a month. * When significant statistical differences were found among the three groups, we examined the statistical difference between each group of two. Patient-reported adverse events in the orlistat-treated groups were all related to the gastrointestinal tract. The most commonly reported events were flatulence with discharge (9.2%), fatty or oily stool with increased defecation (9.2%), feeling of fullness in the stomach (4.6%), and constipation (1.7%). There were no statistically significant differences in side effects between groups A and B. In group A, 11 patients (17.7%) stopped the treatment for the following reasons: cost of the medication (47%), lack of time (33%), or dissatisfaction (20%). In comparison, ten patients (8.9%) in group B stopped the treatment, mainly because of the cost of the treatment (65%) or low motivation (23.5%). The reasons for stopping treatment significantly differed between groups A and B ( p = 0.03). The percentage of patients who attained their weight reduction goals was largest in group A where patients received orlistat and intense follow-up, in addition to a personally-designed diet (Fig. 1 ). Changes in lipid profiles are shown in Table 2 . The treatment resulted in a significant reduction in triglyceride levels in all groups, a significant reduction of low density lipoproteins (LDL) in groups A and B and no significant difference between initial and final high density lipoproteins (HDL) in any group. Figure 1 Weight loss by treatment group* Table 2 Changes in lipid profile during treatment according to treatment group 1 (paired sample t test) Group A Group B Group C Number of patients 2 32 55 28 Initial triglycerides (mg/dl, +/- SD) 170 ± 53 184 ± 49 255 ± 205 Final triglycerides (mg/dl, +/- SD) 139 ± 43 153 ± 35 165 ± 60 Delta (+/- SD) -31 ± 21 -31 ± 25 -90 ± 187 P value (pre-post) <0.001 <0.001 0.01 Initial low density lipids (LDL; mg/dl, +/- SD) 150 ± 30 156 ± 36 152 ± 44 Final LDL (mg/dl, +/- SD) 129 ± 28 143 ± 32 147 ± 34 Delta -21 ± 26 -12 ± 16 -5 ± 34 P value (pre-post) <0.001 <0.001 NS Initial high density lipids (HDL; mg/dl, +/- SD) 42 ± 7.0 44 ± 6.7 47 ± 14.9 Final HDL (mg/dl, +/- SD) 43 ± 6.6 45 ± 6.7 48 ± 15.3 Delta (+/- SD) 0.9 ± 2.9 0.8 ± 3.3 1.4 ± 8.8 P value (pre-post) NS NS NS NS = Not significant. 1 See footnotes to Table 1 2 Patients with pre and post lipid profile analyses Patients in Group A reduced 5.12 kg (range of 5–8 Kg.) of their initial body weight, patients in Group B reduced 7.8 kg (range 10–12 Kg.) from their initial body weight and patients in Group C reduced 3.12 kg (range 5–6 Kg.) of their initial body weight. Discussion Obesity is a worldwide problem. Treatment of a patient for obesity involves two processes: evaluation of the severity of the obesity and general health condition of the patient, and management which includes guidance in how to gradually reduce weight and maintain the new weight together with imparting healthy lifestyle habits and keeping track of improvement. In this study, we examined weight-reduction techniques that can be carried out in the setting of a community family practice. Intense treatment, combining frequent counseling by the family physician and a dietitian with medications (group A), resulted in the best weight reduction and lipid profile improvement in the short period of this study, as was reported earlier in special weight reduction clinics [ 25 - 27 ] The effectiveness of interventions in primary care setting are controversial [ 28 - 30 ]. Beermann et al [ 28 ] in a community survey of 792 patients found that Orlistat was not prescribed according to the approved indication in the majority of cases. The dropout rate was high and most patients had minor gain from the treatment. Linne et al [ 29 ] noted that success rate of Orlistat in primary-care practice is limited by failure to follow prescribing recommendations. A simple questionnaire to 70 patients revealed that in many cases the referral physician had not observed basic rules and regulations, nor given appropriate information on Orlistat use. Hauptman et al [ 30 ] in a study conducted in seventeen primary care centers in the United States. The study indicates that orlistat is an effective adjunct to dietary intervention in the treatment of obesity in primary care settings. There are many advantages to a program involving cooperation between the family physician and the dietitian. The family physician is acquainted with the patient for a longer time than a dietitian. The physician is familiar with the patient's health condition, medications taken by the patient, the patient's environment and lifestyle and can recommend a treatment suitable to the patient's personality and lifestyle. The family physician is knowledgeable about weight-reducing drugs and possible side effects. The patient trusts and has confidence in the family doctor. Together with the dietitian, a personal diet appropriate to the drug treatment can be created. Obesity is a chronic disease. The physician and the patient must recognize that obesity treatment is a prolonged process that extends a lifetime. Since family physicians are usually familiar with their patients as well as with the patient's family and environment for many years, family physicians know what changes the patient can achieve. They can recruit family members to support the necessary lifestyle changes. Also, because of their training, family physicians are the most suitable professional to holistically treat obesity and its complications. There were several limitations in our study. One limitation was the non-random division of patients into groups A, B and C. It is possible that the patients who chose drug treatment differed from those who chose a diet only. Perhaps they were more ready for the weight-reduction process and to expend money for the drug (in Israel, there is a patient co-payment for medications). The rate of women amongst the patients seeking treatment was higher than the rate of men, as in other reports [ 26 , 27 ]. Hence, we assume that our study represented the segment of the population more prone to try dieting and weight-reduction programs. Ways to increase the number of men participating in weight-reduction programs must be found. The study periods for groups A and B (which received orlistat) were shorter than for group C, partly because there is significant co-payment for orlistat and partly because the targeted weight had been achieved earlier. The drug was well tolerated with minimal gastrointestinal side effects. The patients were treated by family physicians that were orientated toward and trained in weight reduction. Results might have been less successful with other physicians. Hence, family physicians should be trained in the subject by increasing both their knowledge and their treatment skills. Healthcare funds must recognize the importance of weight reduction so that they will allocate the necessary additional time and resources of their physicians, clinics and multi-field staff. This study evaluated only the weight reduction period. Long-term results and whether or not the patient maintained the lifestyle change for a long period were not examined. Further studies should examine the best program for maintaining the new weight. In conclusion, this study showed that within the setting of the family practice it is possible to carry out an effective program of weight reduction and achieve significant weight loss. The addition of orlistat can further improve results. We believe that by providing family physicians with the proper tools, similar success can be achieved in many more clinics. Competing interests The author(s) declare that they have no competing interests. Authors' contributions AF and SP conceived and designed the study, participated in the collection, analysis and interpretation of data and drafted the manuscript. SV Participated in the statistical analysis, interpretation of data and draft of the manuscript. MS and EF participated in the design of the study, data collection and interpretetion. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548505.xml
548511	The treatment of migraines and tension-type headaches with intravenous and oral niacin (nicotinic acid): systematic review of the literature	Background Migraine and tension-type headaches impose a tremendous economic drain upon the healthcare system. Intravenous and oral niacin has been employed in the treatment of acute and chronic migraine and tension-type headaches, but its use has not become part of contemporary medicine, nor have there been randomized controlled trials further assessing this novel treatment. We aimed to systematically review the evidence of using intravenous and/or oral niacin as a treatment for migraine headaches, tension-type headaches, and for headaches of other etiologic types. Methods We searched English and non-English language articles in the following databases: MEDLINE (1966–February 2004), AMED (1995–February 2004) and Alt HealthWatch (1990–February 2004). Results Nine articles were found to meet the inclusion criteria and were included in this systematic review. Hypothetical reasons for niacin's effectiveness include its vasodilatory properties, and its ability to improve mitochondrial energy metabolism. Important side effects of niacin include flushing, nausea and fainting. Conclusion Although niacin's mechanisms of action have not been substantiated from controlled clinical trials, this agent may have beneficial effects upon migraine and tension-type headaches. Adequately designed randomized trials are required to determine its clinical implications.	Introduction Migraines and tension-type headaches impose an important burden upon society and the working public. According to the National Headache Foundation, some 45 million Americans suffer from chronic, recurring headaches and 28 million of these suffer from migraine headaches annually [ 1 ]. Furthermore, the work force loses approximately 50 billion dollars per year due to absenteeism and medical expenses caused by headaches, with more than 157 million workdays lost each year to migraine sufferers alone [ 1 ]. Even though advances have been made with regard to the treatment of acute migraine headaches (i.e., the triptan formulations), many patients often discontinue their migraine interventions due to treatment dissatisfaction [ 2 ]. Among individuals seeking treatment for tension-type headaches, the frequency of such headaches is often daily or almost every day [ 3 ]. Unfortunately, chronic tension-type headaches are associated with analgesic abuse [ 4 ], and are difficult to manage in a primary care setting due to frequent comorbid psychiatric or analgesic use problems [ 5 ]. Thus, it is imperative that other methods of treatment be researched and developed in order to increase satisfaction, therapeutic response, and compliance amongst these patients. One novel, but not really new treatment option, is the administration of niacin (nicotinic acid) through intravenous and/or oral routes. Niacin is a well-known over-the-counter (OTC) supplement primarily used for its ability to favorably influence cholesterol levels. Recently, there have been anecdotal reports demonstrating the effectiveness of niacin for aborting acute migraine attacks [ 6 ], and for preventing migraine headaches [ 7 ]. To assess the therapeutic spectrum of niacin's clinical effectiveness, we conducted a systematic review of the literature to examine the evidence of intravenous and/or oral niacin as a treatment for migraine headaches, tension-type headaches, and for headaches of other etiologic types. Methods Literature Search We conducted a systematic search of English and non-English language articles in the following databases: MEDLINE (1966–February 2004), AMED (1995–February 2004) and Alt HealthWatch (1990–February 2004). Articles were searched with the key search terms "Migraine" combined with the Boolean Operator "AND "Niacin" OR "Nicotinic Acid." Additional searches were conducted with the search terms "Headache" and "Tension." To supplement the search, we searched through the references of the articles we found from the databases. Selection of Articles To be included in our final review, articles had to report on the use and administration of niacin for migraine or any other types of headache. We included articles assessing original reports in both peer-reviewed and non-peer-reviewed journals. Quality assessment We determined the evidence grade of each report found, based on the hierarchy of evidence developed by the Oxford Centre for Evidence Based Medicine [ 8 ]. Table 1 displays the hierarchy of evidence. Table 1 Grades of Evidence A Systematic reviews of randomized controlled trials and/or randomized controlled trials. B Systematic reviews of observational studies and/or high-quality observational studies including cohort and case-control studies. C Case-series, case-reports and/or poor quality cohort and case-control studies. D Expert opinion without explicit critical appraisal, or based on physiology, bench research or "first principles." Search Results A total of 14 articles were screened [ 6 , 7 , 9 - 20 ]. Five articles were excluded in total; three because niacin was not the sole therapeutic agent used for the treatment of headache [ 16 ], histaminic cephalgia [ 17 ], and migraine [ 20 ]; and two because the reports were opinion pieces without any objective or subjective data to support the assertions made [ 13 , 19 ]. In total, nine articles were found to meet the inclusion criteria and were included in this systematic review [ 6 , 7 , 9 - 12 , 14 , 15 , 18 ]. Table 2 displays the characteristics of the studies included in this review. Table 2 Summary of Articles Demonstrating Niacin's Effectiveness for the Treatment of Migraine Headaches, Tension-type Headaches, and Headaches of other Etiologies Reference Condition n Protocol Outcome Evidence Grade 9 Migraine headaches 21 One initial intramuscular injection (IM) followed by a series of 6 or 8 intravenous (IV) treatments (maximum 50 mg), then regular IM injections (25–50 mg) combined with 50–150 mg of oral administration. 17 of the 21 subjects had a positive response. C: case-series 10 Headaches of different etiologic types 100 100 mg of IV sodium nicotinate or niacin. 75 of the 100 subjects had complete relief. C: case-series 11 Migraine headaches 15 100 mg of IV niacin, and an additional 50–200 mg if necessary to ensure a flushing response of more than 15-minutes. 13 of the 15 subjects had a positive response. The headaches were relieved in 27 of the 31 times when niacin was administered by IV administration. C: case-series 12 Tension headaches 35 22 subjects received 100–200 mg of IV niacin for a total of 53 times. 13 of the 22 subjects had a positive response. The headaches were relieved in 41 of the 53 times when niacin was administered by IV administration. C: case-series 14 Emotional or tension headaches 5 100 mg of IV niacin regularly for 12 weeks combined with a graded schedule of oral dosing, beginning at 300 mg daily, increasing to 900 mg daily, and tapering down to 300 mg daily. All 5 cases of emotional or tension headaches were very responsive to both IV and oral niacin. C: case-series 15 Tension headaches accompanied with depression 50 100 mg of IV niacin regularly for 23 weeks and then continued once every 2 months and as needed. This was combined with a graded schedule of oral dosing, beginning at 300 mg daily, increasing to 900 mg daily, and tapering down to 300 mg daily. In 44 of the 50 subjects the results with niacin therapy were very satisfactory or favorable. C: case-series 18 Migraine headaches 1 300–500 mg of niacin were chewed and allowed to slightly dissolve in the mouth. Resolution of migraine headaches. C: case-report 6 Migraine Headaches 2 500 mg of oral niacin taken at the onset of acute symptoms. In 2 of the 2 subjects, niacin aborted the acute migraine symptoms. In the first subject, niacin resolved the acute attacks in 4 of 4 occasions. In the other subject, niacin resolved the attack on one occasion. C: case-report 7 Migraine headaches 1 375 mg of oral sustained-release niacin twice daily for 1 month, and 375 mg once daily for 2 months. Migraine-free for the first month, and a marked reduction in migraine headaches over the next 2 months. C: Case-report Limitations Article #1 [ 9 ] This case series of 21 patients was limited as it was uncontrolled and involved a small number of patients. The methods that were used to evaluate efficacy of the treatment were primarily based upon a subjective questionnaire or medical record. The results would have been more meaningful if all the patients used the questionnaire to evaluate their treatment responses. Finally, although the symptomatic changes were witnessed immediately following niacin injection, the lifestyle recommendations may have had therapeutic benefits as well. Article #2 [ 10 ] This study involved 100 patients having headaches of multiple etiologies. This study was not properly controlled, but did at least provide some comparison against a group of patients that were not administered the flush forms of intravenous niacin. The fact that the control group did not substantially benefit from the intravenous niacinamide lends more therapeutic efficacy to the ability of intravenous sodium nicotinate or niacin to have a marked therapeutic effect. The sample size was sufficient in number (n = 100), but the determination of therapeutic effectiveness was purely subjective and did not include any questionnaire or standardized method of evaluation. Side effects were minimal; 2 patients experienced mild abdominal cramps, 1 patient vomited but was suspected as having a pathologic condition of the stomach, and 1 other patient with migraine vomited forty-five minutes after the injection. A few patients found the treatment to be worse than their headaches. The authors concluded that the relief of headaches seemed to be correlated with the degree of flushing from the sodium nicotinate or niacin, and that this therapy was most useful among the migraine, spinal tap, and idiopathic groups. Article #3 [ 11 ] A number of shortcomings were evident in this article. The first of which was that the study was not properly controlled and did not contain a placebo group or a group of patients acting as self-controls. However, some of the patients were given intravenous treatments on more than one occasion. This procedure helped in determining treatment reliability and reproducibility since niacin appeared to achieve therapeutic benefits on several occasions. There were no standardized methods of evaluating efficacy since the treatment responses were based upon the medical record and subjective reports. The results would have been more meaningful if all the patients used a standardized questionnaire prior to and after each treatment. In addition, there was only one male subject and 14 females in the study. Article #4 [ 12 ] In this study of 35 patients were given treatments of intravenous dihydroergotamine methanesulfonate, intravenous niacin, or oral combination tablets of ergotamine tartrate and caffeine. Although no control group was used in this study, the patients were given multiple treatments on several occasions. This offered an interesting comparison to be made between niacin and other treatments. Since no control or placebo group was included, it cannot be determined if the therapeutic results were due, in part, to chance or placebo effects. The results might have been more meaningful if all the patients were given a standardized questionnaire prior to and after each treatment, and if an objective measure was incorporated to further substantiate patient responses. Article #5 [ 14 ] These cases reported were well described and clearly demonstrated therapeutic responses during the intravenous and oral niacin therapy. The results would have been more reliable if a comparison had been made to a control group or to a similar patient cohort that were not given the same treatments. Even if the patients served as self-controls, and were told to stop the niacin treatment for a specified period of time, more information could have been gained from their therapeutic responses to niacin. Overall, this report of five cases provides an interesting approach to patients having chronic tension headaches and depression. Its value is limited by the difficulty in extrapolating these findings to a greater number of patients. Article #6 [ 15 ] In this study involving 50 patients there was no data that listed pertinent identifying information, treatment response, past treatments, and duration of treatment for each patient. This would have strengthened the report by being more descriptive, and thus more amenable to critical analysis. If a comparison were made to a control group or a similar patient cohort not given the same treatments, more validity could be have been ascribed to this method of treatment. The patients could have also served as their own controls, thus providing more information about the therapeutic responses to niacin. It cannot be determined if the therapeutic results were due, in part, to chance or placebo. No method of evaluating efficacy was mentioned, except that the responses to niacin were "very satisfactory" in 44 of 50 cases. The results would have been more meaningful if all the patients were given a standardized questionnaire prior to and after each treatment, and if an objective measure was incorporated to substantiate their responses to niacin. Article #7 [ 18 ] In this report, Hall describes the use of niacin for his migraine headaches remarking that the migraines resolved when intense flushing occurred. According to Hall, niacin's benefits and side effects might be due to its ability to release serotonin and histamine from the stomach. There is no reason to doubt Hall when describing his therapeutic response to niacin. However, his report was brief, had no control and was entirely subjective as he was the participant as well as examiner. Article #8 [ 6 ] In this report of 2 cases, it was found that in both cases migraines were relieved with oral niacin. The report would have been more rigorous if the patients had acted as their own self-controls. The value of the report is further diminished by the difficulty in extrapolating these findings to a greater number of patients. Article #9 [ 7 ] This case report was of a 62-year-old woman with a 40-year history of migraine headaches. The patient never acted as her own self-control, which would have made the findings of the case report more meaningful. The fact that the patient's migraine headaches increased in severity after a reduction in dosage does lend more support to niacin as being a migraine preventive agent. The hypothesized increase in serotonin from niacin administration cannot be proven given the limited amount of data contained in the case report. Like all case reports, its value is diminished by the difficulty in extrapolating these findings to a greater number of patients. Discussion The results of this systematic review indicate that niacin may have a therapeutic effect on migraine headaches, tension-type headaches, and headaches of other etiologies. The quality of the evidence at this point, however, is only hypothesis generating, and randomized trials are required to determine the clinical implications of this novel treatment. There are several important limitations to consider in the interpretation of this review. We did not find any randomized or controlled trials of niacin on these headaches. We cannot determine to what extent publication bias has on the results of this review. We are unable to draw clinical inferences on the results of the included studies as they were of low quality and have a low level of external generalizability. Despite these limitations, we attempted to conduct an exhaustive search and included all reports of relevance. Reasons for niacin's effectiveness can only be considered hypothetical, and require clarification from future randomized controlled trials. In acute migraine headaches some of the symptoms arise from activation of the trigeminovascular complex. Activation of this complex leads to intracranial vasoconstriction causing the migraine aura, followed by headache due to vasodilation of the extracranial vessels and activation of the perivascular nociceptive nerves [ 21 ]. When taken intravenously or orally, niacin causes cutaneous flushing that might abort the acute symptoms of migraine by vasodilating the intracranial vessels, thus preventing the subsequent vasoconstriction of the extracranial vessels. There is evidence that niacin is an effective peripheral vasodilator, but its ability to influence central mechanisms (i.e., cerebral blood flow and cranial hemodynamics) involved in migraine headaches have not been well studied. Niacin causes peripheral vasodilation and cutaneous flushing by inducing the production of prostaglandin D 2 (PGD 2 ) in the skin, leading to a marked increase of its metabolite, 9α, 11β-PGF 2 , in the plasma [ 22 ]. When niacin is administered orally in amounts of 500 mg or topically via a 6-inch patch of 10 -1 M aqueous methylnicotinate on the forearm, PGD 2 is markedly released in the skin and its metabolite appears in high amounts in the plasma [ 22 , 23 ]. It is not known if PGD 2 causes vasodilation of the intracranial arteries, but niacin's ability to abort acute migraine headaches suggests that this might be what is occurring. Old reports cited by Bicknell and Prescott [ 24 ], demonstrate that niacin does indeed cause vasodilation of the cerebral and spinal vessels, and that intravenous administration increases the rate of intracranial blood flow in human beings for 20–60 minutes without any significant change in blood pressure. Unfortunately, there have not been more recent reports examining the effects that niacin has upon cerebral blood flow in human subjects. In terms of tension-type headaches, it appears that intravenous niacin is of benefit acutely due to its presumed central vasodilatory properties. Like migraines, part of the underlying pathophysiology of chronic tension-type headaches involves central mechanisms, such as the trigeminal system [ 26 ]. Chronic tension-type headaches are also associated with cerebrospinal pressure or intracranial venous pressure (or both) [ 26 ]. In fact, tension-type headaches are more similar to migraine headaches than they are dissimilar, in that they seem to progress into migraine headaches due to an escalating pathophysiological process [ 27 ]. Thus, niacin might mitigate the acute phase of tension-type headaches through the same hypothesized mechanism of action described earlier. Some of the reports did demonstrate prophylactic benefits when niacin was administered orally every day. It is now recognized that a deficit of mitochondrial energy metabolism (i.e., impaired mitochondrial phosphorylation potential) plays a role in the pathogenesis of chronic migraine headaches [ 28 ]. Niacin maintains adequate mitochondrial energy metabolism by increasing substrate availability to complex I [ 29 ], and this is how it might function as an effective prophylactic agent for migraine prevention. Two other nutritional agents (riboflavin and coenzyme Q10) augment complex I of the mitochondrial respiratory chain, and have been subjected to clinical trials demonstrating their effectiveness for the prevention of migraine headaches [ 30 - 32 ]. A deficit of mitochondrial energy metabolism may play a role in the pathogenesis of migraine. Since niacin improves mitochondrial energy metabolism by increasing substrate availability to complex I, it might also be an effective agent for migraine prevention. Niacin might also prevent tension-type headaches by improving mitochondrial energy metabolism within the skeletal muscles, and by increasing blood flow and oxygenation to the skeletal muscles. The overall net-effect could be a reduction in lactic acid concentrations, leading to reduced episodes of muscular tension and soreness. Niacin may reduce lactic acid concentrations since supplemental niacinamide (the amide of niacin) has been shown to reduce blood lactate and pyruvate concentrations by more than 50% in a patient with MELAS (mitochondrial encephalopathy, myopathy, lactic acidosis, and stroke-like episodes) syndrome by the third day of treatment [ 33 ]. This possible mechanism might only relate to migraine sufferers, however, since plasma levels of lactic and pyruvic acids were found to be significantly higher in migraine patients compared to patients with tension-type headaches and normal controls [ 34 ]. The side effects of intravenous niacin were found to be minimal from the summarized articles. The most common side effects were abdominal cramping, vomiting, and uncomfortable sensations of the skin and burning. A few of the patients described found the treatments to be worse than their headaches. In the articles where blood pressures were evaluated, little-to-no change occurred among the individuals treated with intravenous niacin. In a more recent study, parenteral niacin given to hypertensive and normotensive patients demonstrated a significant decrease in systolic, diastolic, and pulse pressures among the hypertensive subjects [ 35 ]. With respect to the oral administration of niacin, very few patients reported side effects. Even though niacin was well tolerated orally, we previously reported in a randomized placebo-controlled trial examining the safety of immediate-release or crystalline niacin, that it can be associated with intolerable side effects [ 36 ]. The most common side effects found using 500 mg of immediate-release niacin were unpleasant warmth or flushing, pruritis, chills, tingling, nausea, and vomiting. Approximately 75% of the subjects who were given niacin found it tolerable or difficult to tolerate, but did not indicate that they would never take niacin again. Some 18.2% of the niacin subjects indicated that they found niacin to be intolerable and would never take it again [ 36 ]. By contrast, very few of the patients from the summarized articles discontinued treatment due to the side effects of oral niacin. The side effects of greater concern from oral niacin have to do with sustained- or slow-release preparations. These preparations are better in terms of compliance since patients experience less cutaneous flushing with them. However, the use of these preparations have been associated with reversible hepatic toxicity in doses equal to or greater than 1500 mg per day with an acute onset of clinical symptoms of hepatitis in a relatively short period of time (2 days to 7 weeks) [ 37 ]. Other reports have demonstrated clinical symptoms of hepatitis when much larger doses of sustained-release niacin (greater than or equal to 3 grams per day) were used for months to years [ 38 - 42 ]. Sustained-release preparations have a higher incidence of hepatic toxicity in doses comparable to the immediate-release preparations [ 43 ], but these important differences are not typically mentioned in reviews of niacin's lipid lowering properties. Conclusion Even though niacin's mechanisms of action have not been substantiated from controlled clinical trials. It is possible that this agent has a beneficial effect upon migraine and tension-type headaches and the prophylaxis of these headaches. It is imperative that properly designed controlled trials are developed in order to determine niacin's true therapeutic effects and adverse effect profile. In terms of its ability to abort acute migraine headaches, a placebo-controlled trial of parenteral niacin and sumatriptan seems to be worthy of consideration. In terms of prophylaxis, a placebo-controlled trial of oral niacin and riboflavin or coenzyme Q10 also seems worthy of investigation. Competing interests Dr. Jonathan Prousky is associated with Swiss Herbal Remedies, Ltd., a company that sells nutritional supplements including niacin. They had knowledge of this manuscript and have not seen this manuscript. Authors' contributions JP wrote the manuscript. DS contributed to the text, revised the results section, and assisted with the tables.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548511.xml
543444	Laboratory diagnosis and susceptibility profile of Helicobacter pylori infection in the Philippines	Background Helicobacter pylori diagnosis and susceptibility profile directs the applicability of recommended treatment regimens in our setting. To our knowledge, there is no published data on the culture and local susceptibility pattern of Helicobacter pylori in the Philippines. Methods 52 dyspeptic adult patients undergoing endoscopy from the Outpatient Gastroenterology clinic of the University of the Philippines-Philippine General Hospital underwent multiple gastric biopsy and specimens were submitted for gram stain, culture, antimicrobial sensitivity testing, rapid urease test and histology. Antimicrobial susceptibility testing was done by Epsilometer testing (Etest) method against metronidazole, clarithromycin, amoxicillin, and tetracycline. Results Sixty percent (60%) of the study population was positive for H. pylori infection (mean age of 44 years ± 13), 70% were males. H. pylori culture showed a sensitivity of 45% (95% CI [29.5–62.1]), specificity of 98% (95%CI [81.5–100%]), positive likelihood ratio of 19.93 (95% CI [1.254–317.04]) and a negative likelihood ratio of 0.56 (95% CI [0.406–0.772]). All H. pylori strains isolated were sensitive to metronidazole, clarithromycin, amoxicillin and tetracycline. Conclusion Knowledge of the antibiotic susceptibility patterns in our setting allows us to be more cautious in the choice of first-line agents. Information on antibiotic susceptibility profile plays an important role in empiric antibiotic treatment and management of refractive cases.	Background Helicobacter pylori is a gram-negative bacterium that colonizes the gastric mucosa of more than half of the world's population [ 1 , 2 ] Since its isolation in 1982, the association between H. pylori infection and the subsequent development of chronic active gastritis, peptic ulcer disease, gastric cell carcinoma and B cell MALT lymphoma has been well established[ 3 ]. The principal reservoir of infection is the human stomach and transmission has been epidemiologically linked to person to person contact [ 4 ]. The prevalence of infection is greater in developing countries and is influenced by socioeconomic conditions, ethnic background and age[ 5 , 6 ] In the Philippines, there is scarcity of published data regarding the epidemiology of this bacterium. Locally unpublished reports revealed a prevalence of 5.6% seropositivity rate in children and 60% among 136 adult Filipino patients with dyspepsia using the Clotest ® (Cabahug et. al. 2003 and Caballero et al., 1997, unpublished data ). A lower prevalence rate of 42% was reported by Daez et. al. in 2002 ( unpublished data ) among 375 patients undergoing endoscopy at the Philippine General Hospital utilizing the rapid urease test and histopathology. Microbiological isolation of the organism is the theoretical gold standard for the detection of H. pylori infections. However, isolation of the organism by culture has been highly variable. Success rates depend on the technical expertise of the microbiology laboratory, ranging from 30% to 73%[ 7 , 8 ]. Failure to detect the organism may be due to sampling error, inappropriate transport or culture media and insufficient incubation period. In clinical practice, gastric biopsy with culture is not routinely performed due to the availability of more rapid diagnostic tests in the detection of H. pylori such as urease broth tests, urea breath tests, serologic methods and stool antigen detection. However, the increasing prevalence of resistant strains makes culture and antibiotic sensitivity testing valuable to determine alternative treatment regimens after failure of initial eradication regimen. In the Philippines, due to methodological difficulties in isolating the organism, detection of the organism by culture methods has not been popular. Realizing the increasing prevalence of antimicrobial resistance in other countries and its potential negative impact on the efficacy of many treatment eradication regimens, it is important in clinical practice to determine the prevailing local antibiotic susceptibility patterns when choosing appropriate eradication regimens for H. pylori infections in the empiric setting. This study aims to evaluate the use of culture in the diagnosis of H. pylori infection among patients with dyspepsia, to determine the sensitivity and specificity of culture technique in the detection of H. pylori infection, and to determine the antibiotic susceptibility patterns of H. pylori organisms isolated by culture among Filipino patients. Methods This is a prospective, cross-sectional study involving adult patients with dyspepsia, who had independently been determined to have clinical indications for an endoscopy at the out-patient gastrointestinal clinic of the Philippine General Hospital, a tertiary training university hospital in Manila. Eligible patients were enrolled in the study after informed consent to undergo the required diagnostic testing of endoscopy samples. Patients were excluded if they were less than 18 years old, had a history of proton pump inhibitors (PPI) intake within 2 weeks, H2 antagonists within 1 week and antibiotic intake within 1 month prior to inclusion in the study. The nature and purpose of the study were discussed with the patient until fully understood. All patients with dyspepsia undergoing endoscopy who fulfilled the inclusion criteria had a complete history and physical examination. Data were obtained using a data collection form. Participants underwent upper gut endoscopy as clinically indicated. Pre-procedure preparations for Esophagogastroduodenoscopy were performed according to standard methods. Biopsy of gastric tissue were collected from the antrum and body of the stomach and specimens were sent for (1) histopathologic study, (2) gram staining, (3) culture and sensitivity and (4) rapid urease broth test. Those interpreting results of the above diagnostic tests were blinded. Case Definition A patient with Helicobacter pylori infection was defined as those patients independently assessed by their attending physician based on clinical symptoms and a positive test for any of the two diagnostic tests (histology and rapid urease test). In the evaluation of the diagnostic performance of H. pylori culture, the above clinico-laboratory case definition were used as the comparator reference standard. Description of the Diagnostic Tests Gram stain and Culture Two pieces of gastric tissue were obtained and placed in 0.2 mL sterile saline and transported to the microbiology laboratory for processing. The biopsy specimen was placed in a sterile petri dish and minced with 2 sterile scalpel blades. Specimens were inoculated in both 7% Horse Blood + Brain Heart Infusion Agar (HBAP) and Brucella Blood Agar with Skirrow's supplement (5% defibrinated Sheep's Blood with Trimethoprim (5 mg/L), Vancomycin 10 mg/L and Polymixin B (2500 units/L))[ 9 ] Plates were incubated at 37°C for 7–10 days in a microaerophilic incubation environment and examined every other day (Pack-Microaero, Mitsubishi gas co., Japan). H. pylori colonies are typically small, flat and translucent to grey. On days 4 to 5, all plates with no characteristic colonies, were subcultured to a fresh HBAP to promote growth of slow or fastidious strains and incubated for an additional 3 to 5 days. Plates were examined until the tenth day before reporting a negative growth. Suspected H. pylori colonies were tested for urease, oxidase and catalase production. A modified gram stain was performed on a methanol-fixed smear using crystal violet for 1 minute followed by a water wash and then a safranin counter stain for 30 minutes prior to a final washing with tap water The smear was air-dried and examined under oil immersion. The presence of H. pylori is confirmed by the presence of a gram negative curved bacilli and a positive test for urease, oxidase and catalase production. Five H. pylori isolates that survived the shipping and handling were verified at University of Virginia Center for Studies of Diseases Due to H. pylori . Sensitivity Study Epsilometer test (Etest, AB Biodisk, USA) was used to determine the minimal inhibitory concentrations (MIC). MIC values were read as the intercept of the elliptical zone of inhibition with the graded strip for the Etest. Strains were considered resistant when the MIC was >8 g/ml for metronidazole, >1 g/ml for clarithromycin and >0.5 g/ml for amoxicillin. These breakpoints were used based on the recommendations from the National Committee for Clinical Laboratory Standards (NCCLS) and a large clinical trial [ 10 , 11 ]. For tetracycline, resistance was determined at an MIC of >2 μg/ml based on a previous publications[ 12 , 13 ]. Sensitivity results were compared with a standard susceptible strain of H. pylori (NCTC # 12822) and the University of Virginia Center for Studies of Diseases Due to H. pylori metronidazole resistant culture strain #Cp 2124 and clarithromycin resistant strain #Cp 5535. In the absence of a resistant control for amoxicillin and tetracycline, susceptibility breakpoints set by NCCLS and large clinical trials were used[ 10 , 11 , 13 ]. Rapid Urease Test The Rapid Urease Test (RUT) was performed by placing 0.5 ml of 8% (weight/vol) unbuffered urea in distilled water (pH 6.8) in a clear 0.7 ml Eppendorf tube, to which one drop of 1% phenol red (free acid) suspension was added. The urea solutions was stored at 4°C and prepared on the day of use to ensure color stability. Two gastric biopsy specimens from the antrum and body were placed in the tube. A positive test was indicated by a rapid color change of the media surrounding the biopsy from yellow to magenta followed by a rapid generalized color change throughout the media. A negative result was indicated when there was no change in color appreciated after 2 hours of observation. Histopathologic Examination Specimens were sent to the pathology lab and gastric tissues were fixed and stained with Giemsa and Hematoxylin-Eosin dye. A specimen was read as positive if curve bacilli organisms were seen on microscopy. Pathologists were blinded to the results of the other diagnostic tests. Statistical Analysis Demographic data was described using rates and percentages for categorical variables. For continuous variables, means and standard deviations were used. Measures of accuracy for H. pylori culture were expressed as sensitivity and specificity rates, positive and negative predictive values and likelihood ratios with a 95% confidence interval. Results Patient Characteristics Among 52 patients with dyspepsia, 31 (60%) were positive for H. pylori infection based on the pre-defined case definition. The mean age for H. pylori infected individuals was 44 years ± 13. Seventy percent were males with a male:female ratio of 2:1. The majority of infected patient were married (80%), and had reached only up to the secondary level of education (70%). Fifty-five percent were unemployed. Seventy-five percent of infected patients had access to piped water. No significant differences among the demographic characteristics of H. pylori positive and negative cases were observed. Laboratory Diagnosis of H. pylori All included patients underwent the 3 diagnostic tests for the diagnosis of H. pylori infection (histopathology, rapid urease test (RUT) and culture) (figure 1 ). Fourteen of the 52 patients grew H. pylori on culture. Ten of the 14 positive culture samples were also positive for both histology and RUT, 14 and 10 were positive for RUT and histology alone, respectively. To validate the accuracy rate of H. pylori culture, results of the culture studies were compared with clinically defined cases of H. pylori infection, in this case patients who presents with abdominal symptoms and positive for at least one of the two diagnostic tests, histopathology and RUT. H. pylori culture showed a sensitivity of 45% (95% CI [29.5–62.1%]), specificity of 98% (95%CI [81.5–99.8%]), positive likelihood ratio of 19.93 (95% CI [1.254–317.04]) and a negative likelihood ratio of 0.56 (95% CI [0.406–0.772]). The positive predictive value was 97% (95% CI [74.7–99.7%])) and the negative predictive value was 55% (95% CI [39.8–69.7%]). A total of 14 H. pylori organisms were isolated from 52 clinical specimens. The mean number of incubation time was 3.8 days ± 1 day. All isolates grew on primary plates. All isolates were highly sensitive to amoxicillin (mean MIC of 0.016 ug/ml by Etest)), tetracycline (mean MIC of 0.164 ug/ml SD ± 0.16 SD by Etest), metronidazole (mean MIC of 0.061 ug/ml SD ± 0.04 by Etest) and clarithromycin (mean MIC of 0.016 SD ± 0 by Etest) (table 1 ). Discussion This pilot study reported an H. pylori culture sensitivity rate of 45% and a specificity rate of 98–100% which are comparable to those reported in other countries[ 8 , 14 - 17 ]. High positive predictive values coupled with an intermediate to high likelihood ratio demonstrates that gastric tissue culture is highly specific, making it a useful confirmatory test in the diagnosis of H. pylori infection. Its low sensitivity is acceptable since this method is not recommended as a screening test. This study also supported previous studies that the rapid urease test whether in a gel or liquid preparation is a highly sensitive tool which qualifies as a good screening test among suspected H. pylori infected individuals[ 13 , 18 , 19 ]. Antibiotic resistance has increasingly been recognized as a major cause of treatment failure for H. pylori infection. Primary antimicrobial resistance against clarithromycin and metronidazole is now commonplace in several countries[ 2 , 20 - 26 ]. Regional variations in susceptibility and resistance patterns may be ascribed to differences in local antibiotic prescription practices, antibiotic usage in the community and mass eradication programs for H. pylori infection as part of gastric cancer prevention strategies. These factors may well be expected to influence success of eradication therapy [ 27 - 29 ]. All 14 strains isolated showed sensitivity to all the first line antibiotics namely metronidazole, amoxicillin, clarithromycin and tetracycline. No resistant strains were isolated based on the Etest method. Susceptibility patterns in Europe and the United States revealed that the highest resistance is to metronidazole ranging from 33.1% to 36.9%. Clarithromycin resistance was observed to be 10% in both areas. In contrast, Japanese data showed that clarithromycin resistance was 29% closely followed by metronidazole at 24%. Amoxicillin resistance remained low at 0–1.4% in all three geographic locations[ 3 , 21 , 23 ]. While susceptibility studies were done on large numbers of isolates in foreign data, this local study comprises one of the pioneering attempts, to determine the antibiotic susceptibility pattern of H. pylori infection in the Philippines. Potential reasons for the absence of resistant H. pylori strains in our pilot study point to the type of population enrolled. Based on the selection criteria, these patients had no exposure to previous antibiotic nor had previous H. pylori eradication treatment, a strong risk factor for the development of acquired resistance. Another possible explanation for the low resistance of H. pylori isolates as compared to other Asian countries, is the difficulty procuring antibiotics due to their restrictive cost. In this study, 70% of H. pylori positive patients were unemployed with average incomes below the poverty level, defined as income below the annual per capita poverty threshold of PHP 18,000.00 for the National Capital Region of the Philippines (2001 Philippine Health Situation, Department of Health, Manila, Philippines, ). A study of the World Health Organization's Programme for appropriate Health Care Technology (ATH) has shown a correlation between the occurrence of multi-resistant bacteria and antibiotic consumption patterns. The Philippines has the highest percentage in 1983 of antibiotic utilization among countries (including USA, Japan, United Kingdom) surveyed (>25%). However, majority of the people whether rich or poor allot minimum expenses for medical care at 2.7% and 1.2%, respectively[ 30 ]. Such paradox in resistance patterns may well be explained by the capacity of these patients to actually afford the prescribed duration of antibiotic therapy. While many Filipinos may be (mis-) guided on the appropriate choice of antibiotic therapy by media or product representatives, the cost of these drugs still limits the access to these largely economically disadvantaged group. Although the presence of primary resistance of H. pylori has been well documented in other studies, the absence of primary resistance in our results may also be an underestimate of the true prevalence of H. pylori resistance because of the smaller sample size compared with published literature[ 2 , 20 , 25 , 26 ]. Only with continued surveillance of susceptibility patterns and a larger sample size of isolates will provide a more substantial answer to the issue of resistance of H. pylori in the Philippines. Knowledge of the antibiotic susceptibility patterns in our setting allows us to be more cautious in the choice of first-line agents. The use of culture technique in the diagnosis of H. pylori infection approximates that in published literature abroad. In the absence of standard disk diffusion zone sizes for regimens used in H. pylori eradication regimen except for metronidazole, further establishment of the susceptibility pattern of locally occurring isolates by comparing zone size breakpoints with Etest, agar dilution method and as well as molecular genotyping of resistant strains will be the future direction of this pilot study. Conclusions While the use of culture is not an ideal test for the rapid diagnosis of H. pylori infection, information on antibiotic susceptibility profile plays an important role in empiric antibiotic treatment and management of refractive cases Authors' contributions RVD, LJB, CSA contributed in the microbiologic isolation of H. pylori , manuscript writing and editing EDL, MOD, VD contributed in the specimen processing of biopsy samples, manuscript writing and editing RLG contributed in the research design planning, manuscript content and final editing Table 1 Sensitivity study Helicobacter pylori isolates* ISOLATE NO. AMOXICILLIN TETRACYCLINE METRONIDAZOLE CLARITHROMYCIN Etest Resistance Breakpoints > 0.5 ug/ml > 2 ug/ml > 8 ug/ml > 1 ug/ml 1 0.016 0.032 0.016 0.016 2 0.016 0.19 0.125 0.016 3 0.016 0.016 0.016 0.016 4 0.016 0.38 0.064 0.016 5 0.016 0.38 0.064 0.016 6 0.016 0.19 0.125 0.016 7 0.016 0.032 0.016 0.016 8 0.016 0.38 0.016 0.016 9 0.016 0.032 0.064 0.016 10 0.016 0.19 0.016 0.016 11 0.016 0.38 0.016 0.016 12 0.016 0.032 0.125 0.016 13 0.016 0.032 0.064 0.016 14 0.016 0.032 0.125 0.016 Strain # CP2124 § 0.015 0.015 33 0.015 Strain # CP5535 § 0.015 0.125 1.5 257 all Etest studies done on the Philippine isolates were in the sensitive ranges § UVa (University of Virginia) Metronidazole resistant isolate ¥ UVa Clarithromycin resistant isolate Figure 1 Comparative positivity of diagnostic tests: RUT (rapid urease test) and histology to H. pylori culture . All patients underwent biopsy, RUT and culture studies. Among the 31 clinico-laboratory defined cases, 14 were culture positive. Ten of the H. pylori culture positive cases were also positive for both histology and RUT. All 14 culture positive isolates tested positive for RUT while only 10 were positive for histology.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC543444.xml
423159	A DNA Recombination “Hotspot” in Humans Is Missing in Chimps	null	When Francis Collins and Craig Venter reported the draft sequence of the human genome in 2001, Collins described the so-called book of life as more of a life sciences encyclopedia. In it, we can find our evolutionary history written in the fossil record of our DNA, a parts manual listing the genes and proteins needed to build and operate a human being, and a medical text, gleaned from the genetic variants linked to human disease. Unfortunately, he added, the texts are written in a language “we don't entirely know how to read yet.” Since then, biologists have made great progress in extracting meaning from the human genome. Humans are 99.9% alike genetically, and that 0.1% makes all the difference in terms of appearance, personality, and susceptibility to disease. That 0.1% promises to shed light on the evolutionary forces that control genetic variation as well as the genetic origins of human disease. The TAP2 region harbors a recombination hotspot in humans. What about in chimpanzees: hot or cold? Very small genetic variations—including differences of a single DNA base, called single nucleotide polymorphisms, or SNPs—occur through random mutations. Individuals have two of each chromosome (one from the mother and one from the father), and the combination of SNPs found together on one chromosome can change through the random shuffling of genetic material between the two chromosomes when sperm and egg cells are produced during meiosis. By studying the location and frequency of this reassortment in the genome, biologists hope to understand how recombination affects the overall pattern of SNP variation and how these patterns relate to human disease. An international collaborative effort called the HapMap Project aims to identify the most common SNP associations within chromosomes, known as haplotypes, and then determine which haplotypes are associated with disease. This approach relies on what's known as “linkage disequilibrium”—the nonrandom association of alleles (gene variants) at different locations on a chromosome—to facilitate their search for candidate disease genes. Adjacent SNPs show strong linkage disequilibrium, which means that researchers can select a limited number of SNPs as markers for a haplotype and test their association with disease rather than testing each SNP. Patterns of linkage disequilibrium depend on the rate of recombination—higher recombination rates typically cause less linkage. Demographic factors and chance also affect levels of linkage disequilibrium; while both vary across populations, it has been thought that recombination rates do not. Recombination appears to favor specific genomic regions, termed hotspots, but the observation that the recombinant chromosomes are not passed down in equal proportions suggests that recombination hotspots may be short-lived, appearing as transient blips on the evolutionary radar. Exploring this possibility, Susan Ptak et al. compared a well-studied recombination hotspot in humans, called TAP2, with a similar region in our closest evolutionary cousins, chimpanzees, to see whether they are similarly endowed. Since recombination occurs relatively rarely, researchers have relied largely on indirect methods to determine regional recombination rates. Though recent advances have made sperm analysis in humans more practical (though still technically challenging), such techniques are less feasible with chimps because collecting large amounts of sperm from individual males might compromise their success in mating competition or reduce the genetic diversity of endangered chimp populations. Here, the researchers used an indirect approach to estimate recombination rates from the patterns of linkage distribution, which “reflect the rate and distribution of recombination events in the ancestors of the sample.” They focused on chimps from a single subspecies because the reported high level of genetic differentiation between subspecies could skew estimates of recombination rate variation. Analysis of the TAP2 region revealed 47 SNPs in the human and 57 in the chimp, with an overall lower level of linkage disequilibrium in humans: strong linkage was seen only in adjacent pairs of SNPs in humans, but was found in both adjacent and more distant pairs in the chimps. Using a statistical approach to characterize recombination rate variation between the two species, Ptak et al. found “extremely strong support” for rate variation in humans but found strong evidence against such variation in chimps. Humans and chimps diverged from a common ancestor five to six million years ago and differ at only 1.2% of base pairs on average. That the recombination hotspot does not exist in both species suggests that hotspots are not stable and can evolve fairly quickly. If recombination rates within a small genomic area—at the level of a few thousand bases—can change in such a short time frame between such closely related species, Ptak et al. reason, they may do so within species, too. Such a prospect has important implications for the HapMap Project and disease association studies that rely on linkage disequilibrium. While haplotypes offer a shortcut for identifying candidate disease genes based on typing a certain number of markers, the number of markers required depends on the strength of linkage disequilibrium. If recombination rates differ across human populations, as these results suggest, then the strength of linkage disequilibrium will too—which means that association studies might need to adjust the number of markers needed to flag candidate disease genes in different populations.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC423159.xml
554110	Effects of prostaglandin E2 on the electrical properties of thermally classified neurons in the ventromedial preoptic area of the rat hypothalamus	Background Physiological and morphological evidence suggests that activation of the ventromedial preoptic area of the hypothalamus (VMPO) is an essential component of an intravenous LPS-dependent fever. In response to the endogenous pyrogen prostaglandin E 2 (PGE 2 ), the majority of temperature insensitive neurons in the VMPO show an increase in firing rate, while warm sensitive neurons are inhibited. We have hypothesized that these PGE 2 dependent effects on firing rate are due to changes in the inherent electrical properties of VMPO neurons, which are regulated by the activity of specific ionic currents. Results To characterize the electrical properties of VMPO neurons, whole-cell recordings were made in tissue slices from male Sprague-Dawley rats. Our results indicate that PGE 2 dependent firing rate responses were not the result of changes in resting membrane potential, action potential amplitude and duration, or local synaptic input. However, PGE 2 reduced the input resistance of all VMPO neurons, while increasing the excitability of temperature insensitive neurons and decreasing the excitability of warm sensitive neurons. In addition, the majority of temperature insensitive neurons responded to PGE 2 with an increase in the rate of rise of the depolarizing prepotential that precedes each action potential. This response to PGE 2 was reversed for warm sensitive neurons, in which the prepotential rate of rise decreased. Conclusion We would therefore suggest that PGE 2 is having an effect on the ionic currents that regulate firing rate by controlling how fast membrane potential rises to threshold during the prepotential phase of the action potential.	Background Fever, an elevation in body temperature, is thought to play an adaptive role in the immune system's ability to fight infection [ 1 ]. A suggested mechanism for its production and maintenance is a shifting of the thermostatic set-point into the hyperthermic range [ 1 , 2 ]. Through the integration of both central and afferent thermal information, this set-point is established by the activity of neurons in the preoptic and anterior regions of the hypothalamus (PO/AH) that can be thermally classified on the basis of their inherent ability to respond to changes in temperature [ 3 ]. The majority of PO/AH neurons are considered temperature insensitive, showing little or no temperature dependent changes in firing rate. Approximately 30% of PO/AH neurons can be classified as warm sensitive, responding to local warming with an increase in firing rate [ 4 ]. While there has been considerable debate as to the criteria that should be used to classify a neuron as warm sensitive, we have used a regression coefficient of at least 0.8 impulses·s -1 ·°C -1 . This criterion is based on previous studies that indicate a functional difference for neurons which show this degree of inherent thermosensitivity [ 3 , 4 ]. In addition to responding to local changes in temperature, some of these warm sensitive neurons are also responsive to changes in skin or spinal temperature, while others show thermally dependent changes in their firing rates that may directly correlate with the activation of specific thermoregulatory responses. Although this integrative ability seems to be restricted to warm sensitive neurons in the PO/AH, temperature insensitive neurons may also play an important role in determining the set-point temperature through their synaptic interactions with thermoregulatory effector neurons [ 3 ]. Regardless of thermosensitivity, many PO/AH neurons may respond to adjustments in other homeostatic conditions or the presence of endogenous pyrogens such as prostaglandin E 2 (PGE 2 ), which could shift the thermostatic set-point and alter the activation of thermoregulatory mechanisms [ 2 ]. In response to stimulation of the immune system, changes in the activity of neurons in specific regions of the PO/AH may be responsible for the adjustment of the thermostatic set-point that results in an elevation in body temperature. Physiologic evidence suggests that in response to endotoxins such as lipopolysaccharide (LPS), this shift in set-point is mediated by the activation of afferent neural pathways or the production of systemic pyrogens, which ultimately leads to the local production of PGE 2 within the hypothalamus [ 5 , 6 ]. Early microinjection studies clearly established a role for prostaglandins in the production of a fever and later identified the importance of the region surrounding the OVLT in this response [ 7 - 10 ]. More recently, it has been shown that fever in response to intravenous LPS is dependent on the presence of the PGE 2 producing enzyme cyclooxygenase-2 in the ventromedial preoptic area of the hypothalamus (VMPO) [ 11 ]. In addition, it has now been demonstrated that unlike other regions of the PO/AH, PGE 2 has a selective effect on the firing rates of VMPO neurons, based on thermosensitivity, with PGE 2 increasing the firing rates of temperature insensitive neurons and inhibiting the firing rates of warm sensitive neurons [ 12 ]. Anatomical studies also support the importance of the VMPO in the production of a fever, demonstrating that either the intravenous injection of LPS or microinjection of PGE 2 directly into the VMPO will produce a fever that can be correlated with an increase in the cellular activation of neurons within the VMPO [ 13 , 14 ]. Using a functional criterion for determining the thermosensitivity of hypothalamic neurons, a clear difference in the effects of PGE 2 on the firing rates of VMPO neurons has been demonstrated [ 12 ]. Based on current models of set-point temperature regulation, this PGE 2 dependent increase in the firing rates of temperature insensitive neurons or decrease in the firing rates of warm sensitive neurons could lead to a hyperthermic shift in the thermostatic set-point and production of a fever [ 3 ]. Yet, little is known about the electrical responses by which PGE 2 regulates the firing rates of VMPO neurons. We have hypothesized that these PGE 2 dependent changes in firing rate are not the result of a change in the frequency of synaptic input to these neurons, but a selective effect on specific electrical properties of VMPO neurons. To characterize these responses, whole-cell recordings were made from VMPO neurons in tissue slices from male Sprague-Dawley rats, in response to changes in temperature and PGE 2 . Results For forty two VMPO neurons, temperature sensitivity and PGE 2 dependent changes in firing rate, electrical activity, and the frequency of synaptic input were determined. The majority of these neurons were classified as temperature insensitive (n = 32). The remaining ten neurons were classified as warm sensitive. With respect to thermosensitivity or responses to PGE 2 , there was no specific pattern to the distribution of these neurons throughout the VMPO (Fig. 1 ). Using the cellularly invasive procedure of whole cell recording, the PGE 2 dependent changes in firing rate that were recorded from VMPO neurons were similar to those reported in an earlier extracellular single-unit recording study [ 12 ]. In response to PGE 2 , fourteen temperature insensitive neurons showed significant increases in firing rate. Thirteen of these neurons had thermosensitivities ≤ 0.4 impulses·s·°C -1 . The firing rates of temperature insensitive neurons, having thermosensitivities ≤ 0.4 impulses·s -1. °C -1 , significantly increased in response to PGE 2 , from 5.75 ± 1.31 impulses·s -1 to 6.5 ± 1.31 impulses·s -1 (paired T test, P = 0.04; firing rates at least 10 minutes into the following washout period = 6.37 ± 1.32 impulses·s -1 ). Of the thirteen temperature insensitive neurons with thermosensitivities of 0.41 – 0.79 impulses·s -1. °C -1 , the majority (n = 10) showed little or no change in firing rate in response to PGE 2 . The firing rates of these neurons did not significantly change from a baseline of 8.4 ±1.21 impulses·s -1 (PGE 2 = 8.55 ± 1.24 impulses·s -1 (paired T test, P = 0.38); washout = 8.34 ± 1.24 impulses·s -1 ). In contrast to the responses of temperature insensitive neurons, the majority (n = 8) of VMPO warm sensitive neurons showed a significant decrease in firing rate during perfusion with PGE 2 . The firing rates of warm sensitive neurons significantly decreased from 15.28 ± 4.93 impulses·s -1 to 12.35 ± 4.85 impulses·s -1 in response to PGE 2 (paired T test, P = 0.003; firing rates at least 10 minutes into the following washout period = 12.87 ± 4.12 impulses·s -1 ). Electrical properties All VMPO neurons recorded in this study had resting membrane potentials of -45.0 ± 1.1 mV (n = 42). There was no significant difference between the resting membrane potentials of temperature insensitive neurons (-45.6 ± 1.2 mV; n = 32) and warm sensitive neurons (-43.1 ± 2.4 mV; n = 10). In addition, resting membrane potential did not change in response to PGE 2 and was not responsible for PGE 2 dependent changes in firing rate. The top panels of Figure 2 show the action potential activity of a temperature insensitive neuron during baseline conditions, perfusion with PGE 2 , and the washout period. While the resting membrane potential did not change from a baseline mean of -43.94 mV, firing rate increased 43.4% in response to PGE 2 , from a mean of 5.11 impulses·s -1 to 7.33 impulses·s -1 . The onset of this response occurred several minutes after perfusion with PGE 2 had begun and lasted approximately 15 minutes beyond the point when perfusion with PGE 2 ended. This was typical of all temperature insensitive neurons which had a significant change in firing rate in response to PGE 2 . These neurons showed response latencies of 3.5 ± 0.69 minutes and durations that ranged from 7 to 40 minutes before firing rate returned towards the baseline level. The lower panels of Figure 2 show the action potential activity of a warm sensitive neuron during baseline conditions, perfusion with PGE 2 , and the washout period. While the resting membrane potential did not change from a baseline mean of -51.62 mV, the firing rate of this neuron decreased in response to PGE 2 , from a mean of 10.07 impulses·s -1 to 8.40 impulses·s -1 . The onset of this response occurred several minutes after perfusion with PGE 2 had begun and lasted approximately 25 minutes beyond the point when perfusion with PGE 2 was stopped. Similar changes were recorded for the other warm sensitive neurons that were inhibited by PGE 2 , which showed response latencies of 3.90 ± 0.95 minutes and durations that ranged from 10 to 25 minutes. Throughout the entire length of a recording (46.7 ± 1.8 minutes), the amplitude and duration of action potentials recorded from VMPO neurons slowly decreased by an average of 4.6 mV and 0.16 milliseconds, respectively (n = 42). It is presumed that this was due to minor changes in ionic gradients, resulting from the technique of whole-cell recording. Based on the PGE 2 dependent changes in firing rate reported in this study and previous extracellular recordings [ 12 ], these small changes in action potential amplitude and duration did not affect the ability of VMPO neurons to respond to PGE 2 . There were also no significant changes in the amplitudes or durations of action potentials recorded from temperature insensitive and warm sensitive neurons in response to PGE 2 . The input resistance of VMPO neurons recorded in this study significantly decreased in response to PGE 2 from 367.1 ± 24.5 MΩ. to 339.7 ± 23.7 MΩ (n = 42; paired T test, P = 0.0001). This PGE 2 dependent decrease in resistance was independent of thermosensitivity. Figure 3 shows current-voltage plots from a temperature insensitive neuron (Fig. 3A ) and a warm sensitive neuron (Fig. 3B ). For both of these neurons, input resistance decreased similarly in response to PGE 2 , with a reversal potential at or near the resting membrane potential. During the application of a depolarizing current, VMPO neurons did not show PGE 2 dependent changes in the frequency of action potentials (frequency response). However, the majority of VMPO neurons did show PGE 2 dependent changes in another characteristic of neuronal excitability, the first spike latency. Temperature insensitive neurons with thermosensitivities ≤ 0.4 impulses·s -1. °C -1 showed a significant PGE 2 dependent decrease in the first spike latency, from 7.4 ± 1.2 ms during baseline conditions to 5.2 ± 0.6 ms in response to PGE 2 (n = 18; paired T test, P = 0.008; Figure 4 ). The first spike latency for these temperature insensitive neurons returned to baseline levels during the washout period (7.6 ± 1.4 ms). In contrast, warm sensitive neurons showed a significant increase in this latency response during perfusion with PGE 2 (n = 7), similar to the response of the warm sensitive neuron in Figure 5 . (baseline = 4.9 ± 0.5 ms; PGE 2 = 7.9 ± 1.1 ms (paired T test, P = 0.046); washout = 4.2 ± 0.5 ms). Temperature insensitive neurons with thermal coefficients of 0.41 to 0.79 impulses·s -1. °C -1 showed little change in this measurement of neuronal excitability (n = 8), in response to PGE 2 (baseline = 7.0 ± 1.3 ms; PGE 2 = 6.8 ± 1.1 ms (paired T test, P = 0.69); washout = 6.8 ± 1.1 ms). In Figure 6 , the averaged pre- and post-spike activity of a temperature insensitive neuron (A) and a warm sensitive neuron (B) are shown during baseline conditions and in response to PGE 2 . In a similar manner to the neuron shown in Figure 6A , the majority of temperature insensitive neurons with thermal coefficients ≤ 0.4 impulses·s -1. °C -1 showed a significant increase in the rate of rise of the depolarizing prepotential in response to PGE 2 (n = 12; baseline = 0.46 ± 0.04 mV·ms -1 , PGE 2 = 0.55 ± 0.05 mV·ms -1 ; paired T test, P = 0.01). In contrast, the majority of warm sensitive neurons showed a significant decrease in the rate of rise of the depolarizing prepotential in response to PGE 2 , similar to the response of the neuron in Figure 6B (n = 8; baseline= 0.61 ± 0.08 mV·ms -1 , PGE 2 = 0.50 ± 0.08 mV·ms -1 ; paired T test, P = 0.012). Although small changes in the voltage deflections that occurred at the end of an action potential showed some degree of change in response to PGE 2 , these changes were inconsistent in both temperature insensitive or warm sensitive neurons. Temperature insensitive neurons with thermosensitivities of 0.41 – 0.79 impulses·s -1. °C -1 did not show a significant change in the rate of rise of the depolarizing prepotential in response to PGE 2 . Synaptic input The frequency of post synaptic potentials (PSPs) recorded from VMPO neurons in the localized environment of coronal tissue slices was predominately inhibitory and insensitive to changes in temperature (Table 1 ). In 92% of the recordings, temperature had little or no effect on the frequencies of either inhibitory post synaptic potentials (IPSPs; m = 0.25 ± 0.06 PSPs·s -1. °C -1 ) or excitatory post synaptic potentials (EPSPs; m = 0.23 ± 0.06 PSPs·s -1. °C -1 ). In response to PGE 2 , the frequency of synaptic input recorded from VMPO neurons did not change (Table 1 ). Discussion PGE 2 dependent changes in the firing rates of VMPO neurons As current models for temperature regulation suggest, the integrated responses of hypothalamic warm sensitive neurons and temperature insensitive neurons play an important role achieving and maintaining a discrete set-point for temperature control [ 2 ]. While their importance in thermoregulatory pathways has yet to be determined, in vitro recordings from tissue slices have also identified neurons in the PO/AH that lack spontaneously generated activity (silent neurons), are predominantly driven to produce action potentials by synaptic input (EPSP-driven neurons), or produce action potentials in a bursting pattern [ 2 , 15 ]. However, only prepotential driven warm sensitive and temperature insensitive neurons were identified in the VMPO. In the generation of a fever, current thermoregulatory models of neural networking suggest that either an increase in the activity of temperature insensitive neurons or the inhibition of warm sensitive neurons would shift the set-point to a more hyperthermic temperature [ 2 ]. Through competing synaptic inputs, either of these responses would alter the temperature at which thermoeffector neurons begin to activate thermoregulatory responses. However, previous studies have not been able to show a correlation between the thermosensitivity of hypothalamic neurons and firing rate responses to PGE 2 or other endogenous pyrogens [ 16 - 19 ]. This may be the result of the various criteria that were used to define thermosensitivity in many of these studies, as well as recordings from more general areas of the PO/AH. In a recent study of the extracellular single-unit activity of neurons in the VMPO, a clear thermal distinction in firing rate responses to PGE 2 was reported, with temperature insensitive neurons excited by PGE 2 and warm sensitive neurons inhibited [ 12 ]. In this previous study, a functionally significant criterion was used to define warm sensitivity (m ≥ 0.8 impulses·s -1. °C -1 ) [ 3 , 15 ]. As this criterion provides a method for identifying integrative warm sensitive neurons in the tissue slice preparation, it allows the experimental findings to have a functional significance in modeling thermoregulation in vivo . In the present study, which uses whole-cell recording techniques to record the intracellular properties of VMPO neurons and the same functional criterion for defining warm sensitivity, similar firing rate responses were recorded. As suggested above, either an increase in the firing rates of temperature insensitive neurons or a decrease in the firing rates of warm sensitive neurons could lead to a hyperthermic shift in the thermostatic set-point and a fever. It is also important to note that while the majority of PGE 2 responsive neurons showed a significant recovery in firing rate during the washout period, some did not. This lack of complete recovery may have resulted from responses to PGE 2 that were considerably long in duration, lasting up to 40 minutes, and minor changes in ionic gradients that can occur over time during whole-cell recordings. As mentioned in the methods, all recordings were closely monitored for any changes that would make them unacceptable. Cellular properties of VMPO neurons and responses to PGE 2 In addition to the direct effects of PGE 2 on the electrical properties of VMPO neurons, the frequency of localized synaptic input was characterized. In the present study, we were specifically interested in monitoring firing rate responses to PGE 2 and characterizing the resulting voltage potential changes (i.e., action potentials and membrane potentials). In order to ensure that we did not interrupt the firing rate responses, we did not perform any voltage clamp measurements and did all recordings in current clamp, with current = 0 pA. This prevented any detailed characterization of synaptic potentials, other than frequency. As has been shown in other regions of the PO/AH [ 15 ], the majority of this synaptic input to VMPO neurons was inhibitory and predominantly insensitive to temperature. In response to PGE 2 , the frequency of PSPs recorded from most VMPO neurons either did not change or showed an increase. However, PGE 2 dependent increases in the frequency of PSPs were not recorded from neurons that showed PGE 2 dependent changes in firing rate. This may have resulted from a smaller degree of synaptic influence on firing rate, due to a change in the input resistance [ 20 ]. Additionally, the lack of local synaptic input from warm sensitive neurons would suggest that the axonal projections of these neurons may not terminate within the VMPO, but form efferent projections to other hypothalamic nuclei such as the paraventricular nucleus. As shown in Figure 3 , the input resistance recorded from most of the VMPO neurons decreased in response to PGE 2 , regardless of temperature sensitivity or PGE 2 dependent changes in firing rate. Since this response had little or no influence on firing rate, we would suggest that decreases in resistance resulted from changes in the activity of multiple currents, which continued to maintain equilibrium at or near the resting membrane potential. While we have also suggested that PGE 2 may cause cAMP concentrations to either increase or decrease within these neurons [ 12 , 21 ], the net effect on hyperpolarizing K + currents and depolarizing Na + or Ca ++ currents may still lead to an increase in conductance that does not result in a change in resting membrane potential. Therefore, although these changes in the input resistance may be an important response to PGE 2 , they are not directly responsible for PGE 2 dependent changes in firing rate. In response to a depolarizing current pulse, PGE 2 dependent changes in the excitability of VMPO neurons were recorded. Using two primary measurements of excitability, the frequency response and first spike latency (see: Results & Methods), our recordings indicate that PGE 2 selectively increased the excitability of temperature insensitive neurons, while decreasing the excitability of warm sensitive neurons. Although there was no significant PGE 2 dependent changes in the frequency responses of VMPO neurons, temperature insensitive neurons showed a significant decrease in first spike latency in response to PGE 2 (Fig. 4 ), while warm sensitive neurons showed a significant increase (Fig. 5 ). This would suggest that PGE 2 is having a direct effect of the activity of transient voltage dependent currents, which are responsible for regulating the excitability and rhythmic firing rates of these neurons. While these current pulses were not matched so that they would result in the same level of depolarization, making it difficult to discern which voltage-gated conductances may be responding to PGE 2 , this data does provide support for our finding of PGE 2 dependent changes in the prepotential [ 22 ]. Several transient voltage dependent currents have been identified in hypothalamic neurons which regulate excitability and rhythmic firing rate activity. These include a slow component sodium current and a calcium dependent potassium current (I K, Ca ) in suprachiasmatic neurons [ 23 ], a low voltage activated calcium current and a I K, Ca current in posterior hypothalamic neurons [ 24 ], a non-inactivating potassium current in neurhypophyseal nerve terminals [ 25 ], and a I A current in PO/AH neurons [ 22 ]. Although any or all of these currents may be present in VMPO neurons, only the I A has been implicated in temperature dependent changes in firing rate, through a mechanism in which the thermally dependent inactivation rate of this current influences the rate of rise of the prepotential that precedes each action potential [ 22 ]. In a similar manner, our data suggests that PGE 2 has a direct effect on the firing rates of VMPO neurons through changes in the prepotential, increasing the rate of rise in temperature insensitive neurons, while decreasing the rate of rise of the prepotential in warm sensitive neurons (Fig. 6 ). Therefore, PGE 2 dependent changes in the firing rates of VMPO neurons may also depend on an ability to influence the inactivation rate of the I A type current. Within the VMPO, overlapping expression of EP 3 and EP 4 receptors may provide PGE 2 with the ability to selectively affect the activity of neurons in this region [ 26 ]. Activation of either receptor subtype is known to influence cellular activity through the regulation of intracellular cAMP concentrations, with EP 4 activation leading to an increase in cAMP and EP 3 activation leading to a decrease [ 26 - 28 ]. Support for this mechanism is provided by evidence showing that cAMP plays a role in thermoregulation and more specifically, in the generation of a fever [ 29 ]. With respect to excitability and rhythmic firing rate activity, several studies have demonstrated that cAMP modulates PGE 2 dependent changes the activity of certain potassium currents, including the I A [ 25 , 30 , 31 ]. Therefore, selective activation of either EP 3 or EP 4 receptors may be responsible for the changes in firing rate and electrical activity we have recorded from VMPO neurons. Conclusion In response to intravenous LPS, the local production of PGE 2 within the PO/AH region results in a thermally dependent change in the firing rates of VMPO neurons. Through a direct effect on the rate of rise of the depolarizing prepotential, which is also a determinant of thermosensitivity, PGE 2 increases the firing rates of temperature insensitive neurons, while decreasing the firing rates of warm sensitive neurons in the VMPO. While the results of this study provide a clear focus for future studies into the conductance mechanisms of these responses, it also supports a functional and important role for the VMPO region and both groups of thermally classified neurons in the production of this type of fever. Methods Anterior hypothalamic tissue slices containing the VMPO were prepared from male Sprague-Dawley rats (100 – 150 grams), which were housed under standard conditions and given food and water ad lib . Prior to each recording session, an animal was anesthetized (isoflurane) and sacrificed by quick decapitation, according to procedures approved by the National Science Foundation and the Animal Care and Use Committee of the College of William and Mary. Following removal of the brain, a tissue block of the hypothalamus was cut using a vibratome into 400 μm thick coronal slices. Two or three slices containing the VMPO were then placed in a recording chamber and allowed to equilibrate for 1–2 hours. Tissue slices were continually perfused with pyrogen free artificial cerebral spinal fluid (aCSF), which consisted of (in mM), 124 NaCl, 26 NaHCO 3 , 10 glucose, 5 KCl, 2.4 CaCl 2 , 1.3 MgSO 4 , and 1.24 KH 2 PO 4 . This nutrient medium was oxygenated (95% O 2 – 5% CO 2 ), warmed to a stable temperature of ~36°C by a thermoelectric assembly, and allowed to gravity flow into the chamber at 1 – 1.5 ml· min -1 (chamber volume = 1.2 ml) [ 32 ]. A small thermocouple, positioned in the recording chamber just below the tissue slices, was used to continuously monitor tissue temperature. Tight-seal whole-cell recordings were made using glass microelectrodes with tip inner diameters of ~2 μm (3–5 MΩ), filled with a solution that consisted of (in mM) 130 K-gluconate, 10 EGTA, 2 ATP, 1 MgCl 2 , 1 CaCl, having a pH of 7.2 and an osmolarity of approximately 295 mOsmols/liter. As described previously [ 33 ], a liquid junction potential of 12.0 mV was subtracted from all recorded potentials. All recordings were made using an integrated patch-clamp amplifier (Axopatch 200B, Axon Instruments) and along with temperature, were stored on digital tape for later analysis. Firing rate was continuously recorded as a voltage measurement from a rate/interval monitor and was determined by action potential triggered input from a window discriminator (FHC Inc.). Specific stimulation protocols to measure input resistance and excitability were generated by a computer that was interfaced with the amplifier (pClamp software, Axon Instruments). Acceptable recordings consisted of action potential amplitudes through 0 mV and stable recordings of at least 20 minutes. Using a stereomicroscope, the recording electrode was positioned in the VMPO, which encompasses 700 μm of the hypothalamus just rostral to the suprachiasmatic nucleus. It extends laterally 900 μm from the third ventricle and dorsally 500 μm from the ventral brain surface [ 34 ]. Once a tight seal (> 2 GΩ) was achieved between the electrode and the surface of a neuron, the cell membrane was ruptured by suction, establishing an intracellular recording. When the activity of a neuron was stable for several minutes, temperature in the recording chamber was varied 2–3°C above and below 36°C, by changing the input voltage to the thermoelectric assembly. Neuronal thermosensitivity (impulses· s -1 ·°C -1 ) was characterized by plotting firing rate as a function of temperature to determine the regression coefficient (m) of this plot. As in previous studies [ 12 , 15 , 35 ], warm sensitivity was defined as a regression coefficient of at least 0.8 impulses· s -1 ·°C -1 . All other neurons in this study were defined as temperature insensitive. After thermosensitivity had been characterized, each neuron was tested for its response to PGE 2 . At a stable temperature (~36°C), the perfusion medium was switched to one containing PGE 2 (200 nM or 1 μM, Sigma Chemical Co.). The duration of exposure to PGE 2 ranged from 5 – 15 minutes, with durations of less than 10 minutes occurring only when there was a clear indication of a response. Exposure to PGE 2 was followed by perfusion with aCSF for a washout period of at least 10 minutes. To determine if PGE 2 had a significant effect on firing rate, one-minute segments of stable activity were digitized for comparison (60 Hz; pClamp Software, Axon Instruments). These segments were collected during baseline conditions (just prior to perfusion with PGE 2 ) at the end of perfusion with PGE 2 , and at the end of a 10-minute washout period (or when firing rate returned to baseline levels). For each segment, a mean and standard error were calculated (Sigmaplot software, SPSS Inc.). A significant response to PGE 2 was determined by comparison to the baseline level using a standard T-test (P ≤ 0.05). Changes in resting membrane potential and the action potential waveform in response to PGE 2 were also characterized. As in a previous study [ 32 ], the resting membrane potential was continuously recorded. After rapid changes in membrane potential (including action potentials) were filtered out (half amplitude response setting at 0.5 Hz), one minute segments of voltage activity were digitized (60 Hz) during baseline conditions, perfusion with PGE 2 , and the washout period. To characterize changes in the action potential, an averaged action potential from ten inherently activated action potentials was produced for each of the experimental conditions (sampling rate = 66.7 kHz). Measurements were made of action potential amplitude (resting membrane potential to peak) and duration (at one half peak). Measurements were also made of the rate of rise of the depolarizing prepotential that precedes each action potential [ 22 ]. For resting membrane potential and action potential measurements, significant responses to PGE 2 were determined by comparison to baseline using a standard T-test (P ≤ 0.05). Throughout each recording, input resistance and excitability were measured every 5 – 10 minutes to determine changes in response to PGE 2 and to insure that recordings remained stable. Large changes that did not return towards baseline levels were a characteristic of a deteriorating recording and marked the end of legitimate data. Input resistance was determined by the slope of a current-voltage plot obtained from a computer generated protocol in which a series of ten current pulses (-10 to -100 pA) were administered [ 32 ]. In addition, two primary characteristics of neuronal excitability were measured in response to a depolarizing current [ 36 ]. The frequency response was determined by the number of action potentials produced during the depolarizing current. The first spike latency was also measured and was defined as the duration from the start of the depolarizing current to the peak of the first action potential. All significant responses to PGE 2 were again determined by comparison to baseline using a standard T-test (P ≤ 0.05). The frequencies of synaptic potentials (EPSPs and IPSPs) recorded from each VMPO neuron were determined during baseline conditions, perfusion with PGE 2 , and the washout period. As in previous whole-cell recordings, individual potentials were identified as rapid changes in membrane potential of at least 1 mV greater than background noise [ 15 , 20 ]. For each experimental condition, blind counts of PSPs were made over a 20 second duration to produce frequency averages (PSPs·s -1 ). As with the other measurements, all significant responses to PGE 2 were determined by comparison to baseline using a standard T-test (P ≤ 0.05). In addition, the thermosensitivities of EPSPs and IPSPs were characterized by calculating frequency averages (using the same methods as detailed above) at different temperatures (≥ 3°C range) and plotting the results as a function of temperature [ 15 ]. Once a recording had been completed, a stereomicroscope was used to visually confirm the location of the recording electrode. The ventral edge of the third ventricle was used as a reference to determine the lateral-medial and dorsal-ventral coordinates. The coronal position was also specified by the depth of the electrode from the surface of the tissue slice. In addition, the side of the brain from which the recording was made was identified by preparing the tissue slices with more lateral tissue on the left. Tissue slices were then removed from the recording chamber, fixed in a 10% formalin solution, and sectioned again to a thickness of 50 μm. Sections were then stained with giemsa to identify specific hypothalamic areas so that the location of each recording within VMPO could be reconfirmed [ 12 ] [38]. Authors' contributions HJR, carried out the majority of cellular recordings and data analysis. JDG conceived of the study and participated in its design, coordination and completion. Both authors contributed equally to the drafting of this manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554110.xml
555554	Abdominal surgical site infections: incidence and risk factors at an Iranian teaching hospital	Background Abdominal surgical site infections are among the most common complications of inpatient admissions and have serious consequences for outcomes and costs. Different risk factors may be involved, including age, sex, nutrition and immunity, prophylactic antibiotics, operation type and duration, type of shaving, and secondary infections. This study aimed to determine the risk factors affecting abdominal surgical site infections and their incidence at Imam Khomeini, a major referral teaching hospital in Iran. Methods Patients (n = 802) who had undergone abdominal surgery were studied and the relationships among variables were analyzed by Student's t and Chi-square tests. The subjects were followed for 30 days and by a 20-item questionnaire. Data were collected through pre- and post-operative examinations and telephone follow-ups. Results Of the 802 patients, 139 suffered from SSI (17.4%). In 40.8% of the cases, the wound was dirty infected. The average age for the patients was 46.7 years. The operations were elective in 75.7% of the cases and 24.7% were urgent. The average duration of the operation was 2.24 hours, the average duration of pre-operative hospital stay 4.31 days and the average length of (pre- and post-operation) hospital stay 11.2 days. Three quarters of the cases were shaved 12 hours before the operation. The increased operation time, increased bed stay, electivity of the operation, septicity of the wound, type of incision, the administration of prophylactic antibiotic, type of operation, background disease, and the increased time lapse between shaving and operation all significantly associated with SSI with a p-value less than 0.001. Conclusion In view of the high rate of SSI reported here (17.4% compared with the 14% quoted in literature), this study suggests that by reducing the average operation time to less than 2 hours, the average preoperative stay to 4 days and the overall stay to less than 11 days, and approximating the timing of shaving to the operation and substituting cefazolin for cefaluthin when prophylactic antibiotic is to be administered, the SSI may be reduced to a more acceptable level.	Background SSIs are among the most common hospital acquired infections comprising 14–16 percent of inpatient infections[ 1 ]. SSI is a dangerous condition, a heavy burden on the patient and social health system[ 2 ]. Such infections lengthens bed stay for an average of seven days. Potential sources of infection are the patient (especially contamination by alimentary tract bacteria), hospital environment, food, other patients, staff, infected surgical instruments, dressings, and even drugs and injections [ 3 ]. The incedence of SSIs with regard to abdominal surgical sites and operating conditions is as follows: Clean wounds (1.5–3.7%); clean-contaminated wounds (3–4%); contaminated wounds (8.5%); dirty-infected wounds (28–40%); in laparoscopy (10%) umbilical hernia (2–5%); in the cancer of the colon without taking antimicrobial drugs (30–60%); or with antibiotic and proper intestine wash (10%); in colostomy (above 50%); in colon perforation (20%); in stomach cancer and surgery (20%); in hernionite (50%); in adult appendectomy (10–20%); in children's appendicitis (2–5%); in aged appendicitis and in pregnant women (10–50%); and in AIDS victims (above 50%); in liver abscess (20%); in hydatid cyst (2–5%); in acute and chronic cholecystectomy without stones (10%); in acute septic cholangitis (10–20%); in laparoscopic cholecystectomy (2–5%); and in splenectomy (2–5%) [ 4 ]. SSI is identified with redness, inflammation, heat, pain, a temperature of 38°C, and septic drainage from the surgical site during the 30 days following operation[ 4 ]. Several factors are to be taken into account for SSI, some of which are as follows: While we could not find any significant correlation between sex and SSI rate, age proved to be an important factor; the rate of wound infection for 15 to 24-year-old patients was only10% but increased significantly for those over 65 years of age. The extent of SSI was doubled for obese patients. The duration of surgical operation also proved to be a significant factor: only 3% of operations lasting 30 minutes or less led to infection, while for operations lasting more than 6 hours this rate leapt to 18%. SSI rate increased with longer durations of preoperative bed stay, but preoperative showers with a disinfecting soap such as chlorhexidine or Betadine decreased the cutaneous bacterial load. According to Kruise, the rate of infection was reduced to 1.3% among patients who showered with disinfecting soap containing hexachlorophene. In those who showered with ordinary soap the corresponding figure was 2.1%, and for patients who did not shower at all it increased to 2.3%. However, another study on 5536 subjects showed no decrease in SSI in patients who showered with chlorhexidine preoperatively; the rate was 4% [ 4 ]. Among other factors that delay wound healing or increase the infection rate are cigarette smoking, which increases the postoperative infection rate 5-fold, and the use of steroids, which delays wound healing and increases the infection rate by 9 percentage points from 7% to 16%. Aseptic surgical techniques are claimed to decrease the infection rate, though not to zero [ 3 ]. However, the administration of prophylactic antibiotics 30 to 60 minutes before surgery, decreases the incidence of SSI [ 4 - 10 ]. Obviously, remote infections increase SSI. Other contributing factors are the type of surgery and secondary infections. The object of this study was to assess SSI, and the incedence of the factors contributing to such infections at Imam Khomeini Hospital in Iran. Methods In this study, 802 patients in a teaching hospital during the 15 months from April 2002 to July 2003 underwent abdominal surgery and were studied for SSI and the factors affecting it. Initially, 884 patients were recruited for the study but 82 were excluded on the basis of the following criteria: deficient medical records; patients operated at a different hospital and subsequently transferred to Imam's Hospital: or patients deceased during the operation or within the following 30 days. The dependent variable in this study was abdominal surgical site infection, defined as redness, swelling, pain, temperature above 38°C, during the 30 days after operation. The independent variables were: age, sex, site operated, body mass index, time of shaving the site of incision, administration of prophylactic antibiotics, type of surgical operation, duration of operation, duration of preoperative bed stay; preoperative shower, type of shaving, and accompanying conditions. The data were collected through a 20-item questionnaire. The stages for data collection and information completion were as follows: identification of patients; preoperative interview; postoperative interview; record completion; weekly examinations and telephone follow-ups for 30 days following operation; and pre-discharge examinations. The collected data were analyzed by the SPSS 10 package, using Student's t-test for continuous variables and chi-square test.for categorical variables. To the best of our ability, this study was conducted with due attention to research ethics.however, problems met in the follow-ups the patient's discharge imposed limitations on the study Results Of the 802 patients studied who had undergone abdominal surgery 139 (17.4%) suffered from SSI as defined in Table 1 . No infections were observed in the other 663 cases (82.7%). So far as wound type was concerned, we found clean wounds in 109 cases (13.6%); clean-contaminated wounds in 214 cases (26.7%); contaminated wounds in 307 cases (45.8%); and dirty infected wounds in 112 cases (14%). While 255 cases (28.1%) did not shower before the operation, the other 577 patients (71.9%) did. Table 1 Population Distribution based on the type of operation and SSI Incidence. Type of operation Frequency Percent SSI incidence Percent splenectomy 24 3 -- -- cholecystectomy 272 33.9 25 9.2 umbilical hernia 35 4.4 5 14.3 appendicitis 132 16.5 19 14.4 stomach cancer 58 7.2 23 39.7 Excision biopsy 15 1.9 -- -- laparotomy 142 17.7 25 17.6 cystectomy 9 1.1 4 44.4 colon cancer 24 3 14 58.3 colostomy 15 1.9 10 66.7 laparoscopy 18 2.2 -- -- abdominal mass 10 1.2 2 20 ileostomy 10 1.2 5 50 Intestinal adhesion 5 0.6 4 80 others 33 4.2 4 12.1 total 802 100 140 17.4 The body mass index for 73 patients (9.1%) was above 30, indicating obesity. Over half the patients (403 cases) suffered from accompanying conditions such as diabetes, high arterial blood pressure, kidney or liver failure, malignancy, febrile condition, cardiac disorders, thyroid disorders, blood disease, chronic obstructive pulmonary disease, convulsion, hyperlipidemia, or immunological disorders; or had previously undergone surgical operations. The rest were free of accompanying conditions (Table 2 ). Table 2 SSI Distribution based on the factors involved. Variable Classification SSI (+) SSI (-) total P. Val. N % N % N % Age Age groups under 25 4 3.7 103 96.3 107 100 <0.001 Age groups 25–65 102 18.1 462 81.9 564 100 Age groups above 65 33 25.2 98 74.8 131 100 Sex Female 61 15.1 344 84.9 405 100 <0.093 male 78 19.6 319 80.4 397 100 Type Of Wounds Clean wounds 5 4.6 104 95.4 109 100 <0.001 Clean contaminated wounds 9 4.2 205 95.8 214 100 Contaminated wounds 115 31.3 252 68.7 367 100 Dirty – infected wounds 10 8.9 102 91.1 112 100 Type Of Operation Urgent 29 14.9 166 85.1 195 100 <0.001 elective 110 18.1 497 81.9 607 100 Operation Duration Below 1.5 hours 9 5.4 157 94.6 166 100 <0.001 1.5 – 4 hours 121 19.5 501 80.5 622 100 Above 4 hours 9 64.3 5 35.7 14 100 preoperative bed stay Emergency 24 11.7 181 88.3 205 100 <0.018 1 – 15 days 100 18.6 439 81.4 539 100 More than 15 days 15 25.9 43 74.1 58 100 Shaving Time One hour before operation 29 14.5 171 85.5 200 100 <0.001 12 hours before operation 110 18.3 492 81.7 602 100 showering Before op. Taken 80 13.9 497 86.1 577 100 1.00 none 59 26.2 166 76.8 255 100 Body mass index Under 20 10 17.2 48 82.8 58 100 0.692 Ranging 20.1 – 25 73 16 383 84 456 100 Ranging 25.1 – 30 42 19.5 173 80.5 215 100 Above 30.1 14 19.2 59 80.8 73 100 We found no significant correlations between SSI incidence and sex or preoperative shower. However, correlations with duration of operation, duration of preoperative bed stay, electivity of surgery, lengthening of preoperative shaving time, increasing age, wound infection, site of surgery, type of incision, accompanying disorders, and type of prophylactic antibiotic administered before operation were all significant at p < 0.001. Although differences in SSI rates were not significantly related to BMI, a trend was apparent: SSI rate was higher with low and high BMI. The following prophylactic antibiotics were used: ampicillin, gentamicine, cephalothin, metronidazole, ceftriaxone and cefazoline (table 3 ). Table 3 SSI Distribution based on the type of prophylactic antibiotic administered. variables SSI cefazolin ceftriaxone Metronidazole cephalothin Gentimicine Ampicillin A NA A NA A NA A NA A NA A NA A N % N % N % N % N % N % N % N % N % N % N % N % positive 42 12.1 97 21.3 73 24.5 66 13.1 79 24 60 12.7 19 17 120 17.4 70 30.4 69 12.1 5 50 134 16.9 negative 305 87.9 358 78.7 225 75.5 438 86.9 250 76 413 87.3 93 83 570 82.6 160 69.6 503 87.9 5 50 658 83.1 total 347 100 455 100 298 100 504 100 329 100 473 100 112 100 690 100 230 100 572 100 10 100 792 100 P-Value P = 0.001 P < 0.001 P < 0.001 P = 1.000 P < 0.001 P = 0.018 A = applied NA = not applied Discussion For the 802 participants in this study, the SSI reported was 17.4%, which is well above the 14–16% reported in other studies [ 1 ]. There were particularly high values in cases of umbilical hernia (14.3% compared with previously reported 5%) and stomach cancer (39.7%; in previous studies 20%) [ 4 ]. Increasing age is correlated with greater likelihood of certain chronic conditions, malnutrition and a fall in the body immunological efficiency, causing more extensive SSI [ 4 ]. The present findings supported this argument (p = 0.001). SSI is not correlated with sex [ 5 ], in agreement with previous findings (p = 0.093). The literature shows that SSI increases with obesity, one reason being a decrease in blood circulation in fat tissues [ 7 ]. Malnutrition is another factor predisposing to SSI [ 5 ]. In this study we considered a BMI of above 30 obese and that of below 20 as malnutrition, found no significant correlations between the two ranges and SSI extensity (p = 0.692). But, the previously reported correlation between SSI and pre-operation bed stay (p = 0.018) [ 4 , 5 ] was supported by this study. This is one of the factors to be taken into account. Thus by reducing pre-operation bed stay we may decrease SSI. The findings of this study also proved the risk of SSI to be less in elective surgeries than those referred to emergency departments as cases of acute abdomen, which could result from lack of readiness for operation on the patient's side. Here we should reduce risk factors by preparing the patient for the urgent operation as much as possible. The findings supported the literature by showing that administration of prophylactic antibiotic half an hour before the operation would bring about the best results and the lowest SSI [ 10 ]. This was proved for all antibiotics (p = 0.001) with the exception of cephalothin with (p = 1), which requires a lot more research. The literature shows that with the duration of above 2 hours, the risk of SSI increases [ 4 ]. The average time in this study was 2.24 hours, which must be reduced to below 2 hours although the nature of surgical operations is not always the same. The time of shaving when it approaches the operation and if done by clippers, reduces the SSI risk. In this study the two times: one hour before surgical operations and 12 hours before that were contrasted which supported previous findings with p = 0.001. This is one area where we can lower the risk by approximating the time of shaving as much as possible to that of operation. Other such factors quoted in the literature as the conditions of the operating theatre, personal hygiene, acompanying diseases, immunological disorders, smoking, techniques of surgery, the surgeon's expertise, duration of surgical scrub, preoperative skin preparation, poor hemostasis, failure to obliterate dead space, tissue trauma, and inadequate sterilization of instruments, which were not included in this study might be considered as confounding factors. Conclusion Considering the relatively higher rate of SSI in this study (17.4% compared with the 14% quoted in the literature), especially in such cases as stomach cancers and umbilical hernia, where the rate is considerablly higher, we should carefully reconsider the whole operation procedure. In general, we should do our best to reduce the average operation duration to less than 2 hours and the average preoperative bed stay to less than 4 days. Thus, the present average of 11.2 days for the total bed stay would be reduced to less than 11 days. The time of shaving should approximate the operation time as much as possible. Finally, when the administration of prophylactic antibiotics is required, cefazoline is recommended to substitute cephalothin. Razavy: main researcher (design, analysis, report) Ibrahimpour: (implementation, analysis) Sabouri Kashani: (report, edit) Jafarian: (counsult) Competing interests The author(s) declare that they have no competing interests. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555554.xml
516451	Is HIV-1 RNA dimerization a prerequisite for packaging? Yes, no, probably?	During virus assembly, all retroviruses specifically encapsidate two copies of full-length viral genomic RNA in the form of a non-covalently linked RNA dimer. The absolute conservation of this unique genome structure within the Retroviridae family is strong evidence that a dimerized genome is of critical importance to the viral life cycle. An obvious hypothesis is that retroviruses have evolved to preferentially package two copies of genomic RNA, and that dimerization ensures the proper packaging specificity for such a genome. However, this implies that dimerization must be a prerequisite for genome encapsidation, a notion that has been debated for many years. In this article, we review retroviral RNA dimerization and packaging, highlighting the research that has attempted to dissect the intricate relationship between these two processes in the context of HIV-1, and discuss the therapeutic potential of these putative antiretroviral targets.	Introduction The dimeric feature of the retroviral RNA genome was identified almost forty years ago. However, as with many topics in retrovirology, interest in this area was heightened with the realization that the causative agent of AIDS was a retrovirus. Since then, RNA and protein sequences involved in genome dimerization have been identified for a number of retroviruses, and the dimeric nature of the retroviral genome is known to be important for various critical events in the viral life cycle. These include reverse transcription and recombination, as well as genome encapsidation. To date, a number of informative reviews have been published on retroviral RNA dimerization [ 1 - 3 ], genome packaging [ 3 - 7 ], and the role of nucleocapsid (NC) protein in these activities [ 8 , 9 ]. More recently, a comprehensive review was published that summarized the contributions of in vitro analysis to the identification of retroviral dimerization signals, and provided an overview of the HIV-1 5' untranslated region (UTR) structure with reference to a number of proposed models [ 10 ]. Another, in this issue of Retrovirology , focuses on the different roles of different dimer linkage structures amongst various retroviruses [ 11 ]. In this review, we will focus on results from in vivo studies that provide insights into the relationship between retroviral RNA dimerization and packaging, and the biological relevance of these activities to viral replication. Retroviral RNA dimerization The first evidence for the existence of a dimerized RNA genome came in 1967 when it was shown that viral RNA from each of Rous sarcoma virus (RSV), avian myeloblastosis virus (AMV), murine leukemia virus (MLV), and mouse mammary tumor virus (MTV) displayed sedimentation constants between 64S and 74S in sucrose gradients [ 12 ]. Since these sedimentation constants and corresponding molecular weights were much larger than those of most other known viral RNAs, the structure of these RNA genomes became a matter of great interest. Experiments showing that the 62S RSV RNA species could be converted to a 36S species by heat treatment suggested a disaggregation of the 62S RNA into smaller RNAs, and implied that the fast-sedimenting (62S) RSV RNA was actually an aggregate of smaller (36S) RNAs [ 13 ]. The first real understanding of this putative aggregate RNA structure came in 1975 when RNA from the endogenous feline retrovirus, RD-114, was visualized by electron microscopy (EM), and it was apparent that the 52S RNA molecule existed as an extended single strand that contained a central Y- or T-shaped secondary structure [ 14 ]. It appeared that this 52S molecule actually consisted of two half-size molecules, joined together by the Y- or T-shaped structure, which was termed rabbit ears (RE). It was later shown that the RNA had a poly(A) sequence at each of the two free ends. More importantly, this indicated that nucleotides involved in this RE, or dimer linkage structure (DLS), resided in the 5' region of the RNA genome [ 15 ]. Similar structures were also reported for numerous other type C RNA viruses [ 16 - 21 ]. The absolute conservation of a DLS among retroviruses was strong evidence that the dimerization process must be critical to the retroviral life cycle. With the discovery that the causative agent of AIDS was also a retrovirus, inhibition of RNA dimerization was proposed as a possible therapy for HIV, and HIV-1 RNA dimerization became an intensely studied topic. Both in vivo and in vitro approaches have been used to study retroviral RNA dimerization. The in vivo approach is that whereby RNA is isolated from virions produced in tissue culture and then analyzed by native Northern blotting [ 22 ]. The other method involves synthesis of short segments of viral RNA in vitro , and then studying the ability of these fragments to form dimers. The HIV-1 DLS was originally identified when it was shown that an in vitro -transcribed fragment of HIV-1 RNA could form two major bands on a native gel after incubation at 37°C for 15 min [ 23 ]. The lower band had the expected size of the RNA fragment, while the upper band corresponded to a dimer. In vivo evidence for a role of the NC protein in the dimerization process was already available [ 24 ], and this study also showed that NC could bind to viral RNA and increase the rate of dimerization of the RNA fragments in these in vitro dimerization assays [ 25 ]. It was subsequently reported that an RNA fragment representing nt 1–311 of HIV-1 RNA (Mal strain; a chimera of subtypes A and D) could not only form dimers, but that RNAs containing these first 311 nt could dimerize 10 times faster than RNA sequences at positions 311–415 that were previously shown to be sufficient for HIV-1 RNA dimerization [ 25 ]. Based on these results, the authors concluded that sequences upstream of the splice donor site are involved in the dimerization process, and proposed that sequences in this region somehow hastened the reaction. The key nucleotides involved in this RNA dimerization event make up a palindromic sequence, 274-GUGCAC-279, between the PBS and the major splice donor [ 26 ], and RNA sequences on both sides of this palindrome can form a stem-loop structure with the palindrome in the hairpin loop. Deletion of this stem-loop motif (nt 265–287) completely abolished dimerization of the 1–615 HIV-1 RNA fragment in vitro . The palindromic region was termed the dimerization initiation site (DIS) and it was proposed that this structural element could be exploited for targeted antiviral therapy by antisense oligonucleotides [ 26 ]. These findings were later confirmed when a 19 nt sequence upstream of the 5' major SD was shown to be part of the HIV-1 RNA dimerization domain (Lai strain; subtype B) [ 27 ], and it was found that in vitro dimerization of a 224–402 nt RNA fragment was completely blocked by an antisense oligonucleotide that targeted the palindrome [ 28 ]. This led to a "loop-loop kissing complex" [ 29 ] or "kissing-loop model" [ 27 ] of HIV-1 RNA dimerization, in which the 6 nt palindromes on each of the two monomeric RNA molecules interact through Watson-Crick base-pairing. Purine residues flanking the palindrome were later shown to be intricately involved in this initial interaction [ 30 , 31 ] which is believed to shift the equilibrium toward the formation of dimers, allowing the stems to melt and anneal to their complementary sequences on the other RNA molecule, thus forming the stable extended duplex (Fig. 1 ). This model fits with the idea that immature virions contain a less stable dimer involving only base-pairing of the palindromes, but that the mature virions contain a more stable structure, the extended duplex. Subsequent phylogenetic analysis of over 50 HIV-1, HIV-2, and simian immunodeficiency virus (SIV) nucleotide sequences showed an absolute conservation of a predictable structure similar to the DIS, with the hallmark of the HIV-1 DIS motif being a 6 nt palindrome consisting of either a GCGCGC or a GUGCAC sequence [ 32 , 33 ]. Similar kissing-loop models have also been proposed for a number of other retroviruses [ 34 - 41 ]. Figure 1 HIV-1 5' RNA Structural Elements. Illustration of a working model of the HIV-1 5' UTR showing the various stem-loop structures important for virus replication. These are the TAR element, the poly(A) hairpin, the U5-PBS complex, and stem-loops 1–4 containing the DIS, the major splice donor, the major packaging signal, and the gag start codon, respectively. Nucleotides and numbering correspond to the HIV-1 HXB2 sequence. (Adapted from Clever et al . [73] and Berkhout and van Wamel [136]) Despite ample in vitro evidence supporting the above model of dimer maturation, it was not yet known where or when the RNA dimer was actually formed in vivo . However, native Northern blotting analysis of RNA from two Moloney murine leukemia virus (MuLV) protease-negative (PR - ) mutants displayed dimers that migrated more slowly, and showed lower melting temperatures, than that of wild-type [ 42 ]. It was therefore concluded that PR function is required for RNA maturation in MuLV. Similar experiments with a related virus also suggested that the RNA maturation event required an intact, unsubstituted Cys array within the NC domain [ 42 ]. On the basis of these results, a maturation pathway was proposed for MuLV in which Gag polyprotein molecules assemble into a nascent virion containing an immature dimer. The particle would then be released from the cell, and once Gag is cleaved by PR, NC would act on the immature dimer, converting it to the mature form. Evidence for the role of NC in this dimer maturation process came when in vitro analysis showed that NC could convert the less thermostable dimers to a more stable conformation [ 43 ]. Similar results were obtained by others showing that HIV-1 NC could activate dimerization of a 77–402 nt fragment of HIV-1 Lai RNA, as well as convert an unstable dimer, corresponding to the kissing complex, to a stable one [ 44 ]. Taken together, these thermostability conversions seem to resemble the RNA maturations reported in vivo , and, in agreement with earlier proposals [ 24 ], strongly suggest that NC is responsible for the dimer maturation depicted in Fig. 1 . Subsequent in vivo analysis of a panel of HIV-1 NC mutants showed that Cys-Ser substitution of amino acid residues within the second zinc finger decreased genomic RNA dimerization to the same extent as disruption of the DIS [ 45 ]. This finding confirmed the involvement of NC in the dimerization process, and suggests that the kissing-loop model also applies to the in vivo situation. HIV-1 RNA packaging Why a class of viruses would evolve to have such a unique genomic structure is not entirely clear, but it is speculated that the availability of two copies of the genome would be advantageous for recombination during the complex reverse transcription process, that is key to the retroviral life cycle [ 46 ]. Indeed, the dimeric nature of the genome is thought to be responsible for a high rate of recombination during infection [ 47 - 50 ]. Given that most dimerization signals overlap with known packaging elements, it was naturally assumed that it is the RNA dimer that is specifically recognized for packaging in the case of retroviruses, and that this dimeric feature ensures proper packaging of two copies of genomic RNA. A number of studies have attempted to address this question of a link between dimerization and packaging, but let us first review several aspects of the HIV-1 RNA packaging process. The first studies aimed at identifying the HIV-1 RNA packaging signal found that deletion of RNA sequences between the major splice donor (SD) and the gag coding region (i.e. SL3 and adjacent sequences in Fig. 2 ) decreased the levels of genomic RNA packaged into virions [ 51 - 53 ]. Since these sequences were downstream of the major 5' SD, and therefore would not be found in any spliced viral RNA species, it was plausible that this region could be responsible for the selective packaging of genomic RNAs. Analysis of the putative ψ locus from a variety of retroviruses showed that these sequences had the ability to direct the selective encapsidation of heterologous RNAs to which they had been linked artificially [ 54 - 61 ]. In HIV-1, such autonomous packaging signals were mapped to the regions extending 30–40 nt immediately upstream and downstream of the gag start codon [ 62 ]; however, subsequent studies showed that RNA sequences upstream of the 5' SD site also affected RNA packaging [ 63 ]. It was also known that retroviral encapsidation required trans-acting amino acid sequences in the Gag protein [ 51 , 64 - 68 ], and several groups reported that HIV-1 Gag and NC exhibit specific binding affinity for the HIV-1 ψ site in vitro [ 23 , 69 - 72 ]. These findings, combined with chemical and RNase accessibility mapping, as well as computerized sequence analysis, led to the generation of a model for the HIV-1 ψ site that comprised four independent stem-loops [ 73 ] (SL1-4 in Fig. 2 ). Three of these hypothetical stem-loop structures were each shown to serve as independent Gag binding sites, and were proposed to contribute individually to overall packaging efficiency. SL1, SL3, and SL4 were later shown to be critical for packaging specificity in vivo [ 74 , 75 ]. Subsequent in vitro analysis from another group demonstrated that the major packaging signal is an extended bulged stem-loop whose RNA conformation is altered upon interaction with Gag [ 76 ]. However, more recent work indicates that SL2 and SL3 display much higher affinities for NC than SL1 and SL4 in vitro [ 77 , 78 ]. Based on these findings, a model has been proposed to represent the initial complex formed between the NC domains of assembling Gag molecules and the dimeric ψ region [ 79 ]. In this model, SL1 is shown to form an RNA duplex between the two stands, while SL4, instead of directly binding to Gag, contributes additional RNA-RNA interactions that stabilize the tertiary structure of the ψ element. The RNA conformation resulting from this folding pattern is thought to expose SL2 and SL3 for high-affinity binding to Gag. Figure 2 The Kissing-Loop Model of HIV-1 RNA Dimerization. HIV-1 RNA dimerization is initiated by a Watson-Crick base-pairing interaction between two palindromes in the loops of SL1 on two monomeric genomic RNAs. This interaction forms the loose unstable kissing-loop complex. Coincident with virus particle maturation, this unstable dimer is rearranged to form a more stable extended duplex that involves a mechanism whereby the base-pairs in the stems melt and then re-anneal to their complementary sequences on the opposite strand. Nucleotides and numbering correspond to the HIV-1 HXB2 sequence. (Adapted from Skripkin et al. [26] and Laughrea and Jetté [27]) Despite the clear results obtained from simplified in vitro studies such as those mentioned above, the SL1-4 region alone is not sufficient to target RNA into HIV-1 virions in vivo [ 80 ], and the minimal region required to confer autonomous packaging activity actually maps to a larger region covering the first 350–400 nt of the genome, including ≈ 240 nt upstream of SL1 [ 81 - 84 ]. In agreement with these studies, mutations that alter the stability of the poly(A) hairpin stem region, or delete the upper part of the hairpin, severely inhibited HIV-1 replication [ 85 ]. And, these deficits in replication correlated with reduced RNA packaging levels in virions, suggesting that the formation of the poly(A) hairpin is necessary for normal packaging of viral genomes. Subsequent research confirmed the importance of the poly(A) hairpin in the RNA packaging process [ 86 ], and it was shown that similar disruption of base-pairing in the stem of the TAR element also caused profound defects in packaging [ 81 , 86 ]. Finally, deletion analyses of RNA sequences between the poly(A) hairpin and SL1 suggested that unspecified sequences within the U5-PBS region also contribute to HIV-1 RNA packaging [ 83 , 86 ]. Our group later showed that GU-rich sequences in the lower stems of the poly(A) hairpin and the U5-PBS complex contribute to both dimerization and packaging [ 87 ]. In summary, all of the seven predicted stem-loop structures in the HIV-1 5' UTR (Fig. 2 ) are known to be important for genome encapsidation, and all of these RNA structural elements have also been assigned other functions in various steps of the viral life cycle, e.g. the role of SL1 in the initiation of dimerization. The existence of such overlapping functions for these RNA structures raises the possibility that some of these functions, such as dimerization and packaging, might be linked. The evidence for and against the existence of such a link in HIV-1 will be the main focus of the remainder of this review. Is dimerization a prerequisite for packaging? One of the first electron microscopy studies of a retroviral DLS in 1976 proposed that this region "could have some role in packaging the RNA in the virus" [ 16 ]. This raised the question of a possible link between dimerization and packaging that is still debated. The answer to this question has significance in our basic understanding of the retroviral life cycle and may also have implications for therapy, since many groups are actively studying these two activities as potential drug targets. Clues from in vitro studies Early reports on in vitro dimerization of HIV-1 RNA showed that the DLS localized to a stretch of genomic RNA downstream of the 5' SD (nt 311–415) [ 23 , 88 ], and it was noted that this dimerization domain encompassed a previously identified packaging element that had also been shown to bind NC [ 51 - 53 ]. This dependence of HIV-1 RNA dimerization on cis elements required for packaging was immediately interpreted to mean that retroviral RNA dimerization, activated by either NC or Gag precursors, should direct genomic RNA into the virion, implying that dimerization might be a prerequisite for packaging. Since HIV-1, MuLV, and RSV all contain elements involved in dimerization that were also required for packaging [ 23 , 89 , 90 ], it was proposed that dimerization might function as a molecular switch that negatively regulates translation and positively regulates encapsidation [ 88 ]. The existence of a DLS downstream of the major splice donor would seemingly supply a convenient mechanism whereby only genome length RNA would be able to dimerize and subsequently become encapsidated into the virion. However, evidence questioning such a dimerization-mediated mechanism of genomic RNA packaging came from studies showing that sequences upstream of the SD site had even greater dimerization capabilities than those located downstream [ 25 - 27 ]. The involvement of such sequences (e.g. the DIS, SL1) in the dimerization process questioned the link between dimerization and packaging, because these sequences are also found in all HIV-1 spliced viral RNAs. Observations from in vivo studies Early in vivo studies analyzing the structure of virion-associated RNA from rapid-harvest avian retroviruses showed that viral RNA appeared to be a mixture of monomers and dimers [ 91 - 93 ]. Similar results had also been reported with PR [ 94 , 95 ] and NC [ 24 , 94 , 96 , 97 ] mutants, which argued against the notion that dimerization is a prerequisite for packaging. However, analysis of rapid-harvest virus in MuLV showed that genomic RNA was already in the form of a dimer shortly after budding, albeit as a less stable, physically different RNA dimer than that present in mature virions [ 42 ]. Based on these observations it was proposed that MuLV particles never package monomeric RNAs, but rather that the dimeric RNA structure might be integral to the packaging signal that is recognized by Gag during assembly. It was also speculated that the previously reported presence of monomers in viral RNA preparations had resulted from the physical dissociation of fragile unstable dimers during RNA preparation. Similar experiments performed on PR - mutants of HIV-1 showed that substantial amounts of monomeric RNA could be detected [ 98 ]. Since PR - dimers were shown to be less stable than wild-type dimers, it was assumed that dimers were preferentially packaged in PR - particles, but that some fragile dimeric structures had dissociated during RNA preparation. Based on these in vivo results with both MuLV and HIV-1, it was concluded that dimerization is a prerequisite for packaging and should be considered to be a general feature of retrovirus assembly. Further insights into this topic can be obtained by examination of results from a number of studies aimed at understanding the role of the DIS in HIV-1 replication. One such study, in which DIS loop palindrome sequences were mutated, found that mutation of the palindrome to shorter or longer versions of GC stretches did not have major effects on viral RNA dimerization; however, partial RNA packaging defects were observed that also corresponded to diminutions in viral replication [ 33 ]. Based on these data, it was proposed that these DIS loop mutants might have experienced a partial dimerization defect that caused inefficient packaging [ 33 ]. In a similar study, mutation of the palindrome, as well as deletion of the upper stem-loop of SL1 caused drastic reductions in viral infectivity and decreases in both dimerization and packaging of HIV-1 genomic RNA [ 32 ]. In an attempt to explain how these mutations could affect both activities, a model was proposed in which Gag does not specifically recognize the dimerized genome but rather initially interacts with one molecule of genomic RNA that happens to be linked (dimerized) to a second such molecule. Then, during packaging, Gag would effectively bind to two genomic RNA molecules at once. Hence, defects in dimerization would result in subsequent packaging defects. Based on these data, it was also concluded that the encapsidation and dimerization processes are coupled to some extent. Although several groups had attempted to delineate the relationship between dimerization and packaging, the fact remains that the RNA signals that are important for both of these activities overlap in most retroviral genomes; this makes it difficult to interpret the results of mutagenesis studies. In an attempt to generate viruses that would be expected to display selective defects in dimerization or packaging, one group designed a panel of constructs containing mutations in SL1, SL3, or both [ 99 ]. Results from this study showed that deletion of either SL1 alone, or SL3 plus adjacent flanking sequences, reduced genomic packaging, while deletion of SL1 and SL3 simultaneously caused an even further reduction. With respect to dimerization, complete deletion of SL1, or even disruption of the base-pairing in the upper stem, resulted in elevated levels of monomer-sized RNA species on native Northern blots, again confirming the importance of this region for the in vivo HIV-1 RNA dimerization process. Yet, these mutant genomes could still be packaged, suggesting that HIV-1 RNAs need not be dimers for this to happen. Thus, the authors concluded that dimerization is not a prerequisite for packaging but rather serves an independent function in the retroviral life cycle. In the above-cited article, the effects of SL3 mutations on dimerization were not studied, but our group later showed that viruses containing even minor substitutions in or around SL3 could have significant effects on both dimerization and packaging [ 100 , 101 ]. In summary, the in vivo studies described above commonly observed that mutations in 5' RNA sequences affected both dimerization and packaging, presumably due to the close proximity of the RNA dimerization and packaging signals. Can monomers be packaged? In an attempt to separate the dimerization and packaging functions, and to characterize the DIS-DLS region without altering packaging activity, one group generated mutant constructs carrying a duplication of approximately 1000 nt from the HIV-1 5' region (termed E/DLS) including the encapsidation signal and the DIS-DLS [ 102 ]. They found that the presence of an ectopic E/DLS near the 3' region of the genome resulted in the appearance of monomeric RNA in virus particles, suggesting that monomers can be packaged and that dimerization of HIV-1 genomic RNA is not required for packaging. However, they also found that two intact E/DLS regions had to be present on the same RNA molecule in order for packaging of monomers to occur. Therefore, it was assumed that these monomers had been generated from an intramolecular interaction between the two E/DLS regions. If we assume that such an intramolecular interaction between two DLS structures would occur on a single RNA molecule, however, might such a structure then not also appear as a dimer to a Gag protein that was attempting to package it? Although these data were interpreted to mean that dimerization is not required for packaging, they also suggest that some structure that is generated by the interaction of the two E/DLS regions might be recognized by Gag in order to facilitate packaging. In the context of wild-type genomic RNA containing only one E/DLS region, such a structure might then only be generated by an intermolecular interaction between two RNA molecules, i.e. a dimer. Hence, these results also imply that dimerization might be required for proper packaging. In a follow-up study, the same group created mutant HIV-1 particles that contained only monomeric RNAs, and concluded that these mutants demonstrated the complete separation of encapsidation from physical dimerization of retroviral RNA [ 103 ]. However, they also reported that these viruses packaged only monomers, and that packaging efficiencies were approximately half those of wild-type, implying that dimerization is the sole mechanism to ensure the packaging of two copies of viral genomic RNA into each virus particle. In addition, the packaged monomers might have originally been weak dimers that dissociated during extraction and analysis, as has been pointed out in previous reports [ 42 , 102 ]. However, the above results do raise the issue of packaging specificity in mutant viruses. We and others have shown that, in COS cells, HIV-1 can incorporate significant amounts of spliced viral RNA when proper packaging of full-length viral genomic RNA is reduced [ 99 , 104 ]. During assembly, Gag will always successfully package some RNA, and it is important to know the degree of specificity with which monomers versus dimers are packaged. If monomer-packaging mutants concomitantly package high levels of spliced viral RNA, then it is likely that packaging specificity may have been compromised by the existence of an extra E/DLS, and that the packaging of the monomers was non-specific. However, a lack of spliced viral RNA in these virions would indicate that the monomers were packaged with a high degree of specificity, and would have implications as to whether or not Gag initially recognizes viral genomic RNA in a dimeric versus a monomeric state. None of the viruses engineered to package only monomers were able to efficiently establish a new round of infection, suggesting that dimerization is required for replication if not for packaging. It is difficult to predict what other effects the addition of large segments of highly structured RNA might have on the viral life cycle. Another group reported similar phenotypes in the context of an HIV-1 mutant that was designed to have altered Gag/Gag-Pol ratios [ 105 ]. Analysis of virion-derived genomic RNA from these viruses showed an increase in packaging of monomers, demonstrating that stable RNA dimers are not required for encapsidation of HIV-1 genomic RNA. Interestingly, these viruses also showed drastically reduced infectivity. Insights from forced evolution studies We have also been studying the HIV-1 5' UTR and its putative interactions with Gag, and how these interactions affect dimerization and packaging activities. The DIS is known to be important for viral replication [ 32 , 33 , 63 , 99 , 106 - 109 ], reverse transcription [ 47 , 48 , 107 , 109 ], RNA dimerization [ 32 , 99 , 106 , 109 - 111 ], and packaging [ 32 , 33 , 74 , 99 , 107 , 108 , 110 ], as well as packaging specificity [ 99 ]. However, despite the obvious importance of this stem-loop structure, work from our group has shown that defective viral replication caused by deletions in the DIS can be largely corrected by a series of compensatory point mutations identified in matrix, capsid, p2, and NC [ 112 - 114 ]. These findings imply that the RNA sequences comprising the DIS interact in some way with these domains of Gag, and that when the RNA sequences are mutated, the virus will acquire adaptive mutations that potentially restore putative RNA-protein interactions over long-term culture. Since the originally deleted RNA sequences were in the DIS, we had naturally assumed that the major defect of these mutants would relate to RNA dimerization, and that compensatory mutations had arisen to correct defective RNA dimerization activity. To our surprise, this was not the case. Although our mutants did indeed yield reduced levels of dimerized genomic RNA in virus particles, the compensatory mutations in Gag that restored replication capacity [ 112 - 114 ] did not correct dimerization defects [ 109 ]. Rather, compensatory mutations apparently resulted in increased overall levels of viral genomic RNA that were packaged into virus particles, irrespective of impaired RNA dimerization. Similar effects on packaging were observed in the context of compensatory mutations identified during long-term culture of viruses containing mutations outside the DIS, such as the poly(A) hairpin and the U5-PBS complex [ 87 ], and between the PBS and SL1 [ 115 ]. These findings again question the link between dimerization and packaging, since our compensatory point mutations were able to increase RNA packaging levels without correcting dimerization. One possibility is that the revertant viruses somehow gained the ability to package wild-type levels of RNA without correcting dimerization defects, i.e. they packaged more monomers. However, we also cannot rule out the possibility that our point mutations in Gag may have restored weak dimerization properties to the mutated RNAs, and that the latter dimers dissociated during extraction and analysis. In a follow-up study, we created two other DIS deletions and combined them with various combinations of the previously identified compensatory point mutations. We showed that these mutant viruses, ΔLoop (lacking the loop region of SL1) and ΔDIS (lacking the complete SL1) displayed defects in replication, RNA dimerization, and packaging. Once more, all of these but dimerization were largely corrected by the compensatory point mutations in Gag [ 104 ]. Even a virus that lacked the DIS, e.g. ΔDIS, and which never showed any signs of viral growth in tissue culture, was able to replicate to significant extent when it also possessed the compensatory mutations. The mechanism(s) whereby these compensatory point mutations functioned to restore replication had eluded us for some time. Recently, however, we employed an RNase protection assay to discriminate between genomic and spliced viral RNA packaged into virus particles. Our results showed that all of our 5' UTR mutant viruses aberrantly packaged increased levels of spliced viral RNA compared to wild-type virions. More importantly, however, the effect of one of our compensatory point mutations (i.e. MP2; a Thr->Ile substitution at position 12 of the SP1 spacer peptide in Gag) was to exclude spliced viral RNA from being packaged into mutant virions [ 104 ]. Surprisingly, this single point mutation was also able to restore significant levels of virus replication to our ΔDIS mutant virus, which had been noninfectious in both T cell lines and blood mononuclear cells. Previous work had suggested that the packaging of spliced viral RNA is a mechanism used by packaging mutants to fill the space that would normally be occupied by genomic RNA [ 99 ]. Were this the case, then the MP2-mediated exclusion of spliced viral RNA from the virus particle should have been accompanied by increased packaging of genomic RNA. In the absence of MP2, the mutant particles contained lower levels of genomic RNA and higher levels of spliced viral RNA packaged than wild-type. In contrast, the presence of MP2 led to the exclusion of spliced viral RNA, but had no effect on packaging of genomic RNA. In the context of dimerization and packaging in the mutated viruses, it is possible that spliced viral RNAs, which do contain some RNA elements involved in RNA dimerization, including the DIS, might form heterodimers with molecules of genomic RNA. These putative heterodimers might be packageable, but it is unlikely that virions containing such genomes would be able to replicate, e.g. the noninfectious ΔDIS mutant. However, in the presence of MP2, the modified Gag protein might in some way block the formation of such an RNA heterodimer, thereby increasing the probability that dimers form between two genomic RNA molecules, resulting in partially restored levels of virus replication. Since these genomic RNA molecules are already mutated in dimerization signals, these weaker dimers would probably appear on a gel as monomers. In such a model, MP2 would act to restore dimerization, resulting in increased replication capacity, suggesting that dimerization is required for proper packaging to ensure that a particle is infectious. Unfortunately, this is virtually impossible to prove with current in vitro and in vivo protocols. New approaches to study dimerization and packaging within the cell will hopefully allow new hypotheses to be tested. The packaging of spliced viral RNA and/or the exclusion of such RNA species raises the question of whether the viral RNA sequence, or possibly the RNA structure, is important in proper assembly and/or structural integrity of the virus particle itself. Evidence in support of this possibility comes from studies on the binding of NC, in the context of full-length Gag, to viral genomic RNA. This might concentrate Gag proteins onto one or more RNA molecules, thereby facilitating Gag-Gag multimerization in a template-driven manner. Hence, viral genomic RNA would be a structural element, or scaffold, on which the virion can assemble [ 116 ]. Other reports have shown that viral RNA can affect particle morphogenesis [ 116 - 119 ] and structural stability [ 120 , 121 ], although the mechanisms involved are unclear. If RNA structure, or even the dimeric versus monomeric state of the RNA, truly does play a role in virion assembly and/or stability, this might also explain the apparent detection of monomeric RNA in the HIV-1 mutants mentioned above. For example, the duplication of large E/DLS sequences would undoubtedly have altered the overall structure of viral RNA, which might have resulted in the formation of unstable virus particles [ 102 , 103 ]. Degradation of such particles could have indirectly caused the dissociation of dimers that would then appear as monomers on a gel. The fact that these viruses were all noninfectious may also have been due to the formation of unstable virus particles. Consistent with this concept, we found by electron microscopy that HIV-1 mutants lacking DIS stem sequences displayed an increased proportion of immature virus particles [ 114 ]. This might mean that either the RNA structure, or the lack of a properly formed dimer, resulted in the production of virus particles with abnormal morphology. Since RNA can affect Gag cleavage, it is possible that mutations in the RNA might have also compromised the cleavage of Gag precursor proteins, which may subsequently have affected particle maturation [ 122 ]. We believe that proper RNA dimerization may be a prerequisite for efficient virion assembly and structural stability. As stated, the link between dimerization and packaging is a subject of ongoing debate [ 32 , 33 , 42 , 98 , 99 , 102 , 103 , 109 , 110 ], but we and others view dimerization as a prerequisite for packaging. Genomic RNA can be packaged as monomers [ 99 , 102 , 103 , 105 , 109 ], or alternatively as weak dimers that appear as monomers on gels, but mutant viruses that exhibit dimerization defects generally do not grow as well as wild-type viruses. The fact that our ΔDIS-MP2 virus can replicate in tissue culture, despite being severely compromised in genome dimerization, is evidence that efficient dimerization is not required for packaging or replication. In the absence of an authentic DIS, other sequences that affect dimerization may form a weak dimer that allows RNA to be recognized and adequately packaged [ 87 , 100 , 101 ]. The contribution of the DIS might then be to significantly increase the efficiency of the dimerization process, resulting in more efficient packaging and replication. In conclusion, we agree with opinions expressed by others that the generation of virus particles able to package monomeric genomes is possible, but that dimerization is likely to be a prerequisite for the production of infectious viral progeny [ 10 ]. The DIS as a therapeutic target? It is clear that virus replication capacity is significantly affected whenever dimerization and/or packaging are compromised, suggesting that these activities can be exploited as anti-HIV drug targets. Indeed, the DIS was first proposed to be a potential therapeutic target at least 10 years ago, and antisense molecules were directed at this region of viral RNA [ 26 , 123 ], without practical outcome. Other approaches directly target the HIV-1 kissing-loop complex, which resembles the eubacterial 16S ribosomal aminoacyl-tRNA site, i.e. the target of aminoglycoside antibiotics such as paramycin and neomycin [ 124 ], both of which specifically bind to the kissing-loop complex. Drugs based on antibiotics with high affinity and specificity for the DIS may be a worthwhile approach, although efficacy might be compromised by the fact that HIV can replicate in the face of mutations that decrease genomic dimerization by more than 50% [ 104 ]. RNA interference (RNAi) is a novel mechanism that regulates gene expression in which small interfering RNAs direct the targeted degradation of RNA in a sequence-specific manner (reviewed in Lee and Rossi [ 125 ]). Although RNAi is a powerful tool, it is not yet clear whether its therapeutic potential will materialize. This not-withstanding, several reports show that specific degradation of HIV-1 RNA is possible in infected cells [ 125 ], and reductions of p24 levels by as much as 4 logs have been achieved using RNAi directed against HIV-1 tat and rev [ 126 ]. DNA vectors are currently being engineered that will allow for long-term production of siRNAs for use against chronic diseases, such as HIV-1. The DIS might also be a good candidate for sequence-specific targeting of HIV by RNAi therapy since it is highly conserved among naturally occurring virus isolates, and, due to its position upstream of the major splice donor, is contained in all HIV-1 RNA transcripts, both spliced and unspliced. Effective DIS-directed degradation of HIV RNA should confer the same viral phenotype as observed with our ΔDIS mutant, which never showed signs of virus replication in either permissive T cell lines or blood mononuclear cells [ 104 ]. One concern with use of RNAi is how accessible certain RNA sequences might be. For example, complex secondary structures might cause some sequences to be buried and therefore inaccessible to the siRNA. However, this would not be a concern with DIS-directed RNAi, since the DIS contains a 6 nt palindromic sequence that is believed to initiate the dimerization process by binding to an identical sequence on another molecule of genomic RNA. If two 6 nt stretches of RNA can find each other on two 9200 nt strands of highly structured RNA, they should also be accessible to siRNAs. Recently, the practicality of RNAi-based therapies against HIV-1 was called into question when it was shown that HIV-1 was able to escape the antiviral pressure of RNAi by generating substitutions or even deletions within RNAi target sequences [ 127 , 128 ]. This again highlights the versatility and plasticity of the HIV-1 genome. However, in these studies, the RNAi target sequences were located within the tat and nef genes, and the mutations that were generated blocked the effects of the RNAi without conferring any major detriment to virus replication. In contrast, RNAi may be more useful if targeted to more critical RNA elements within the genome, such as the DIS or the Ψ region, since any escape mutations that occur might result in viruses with severely impaired replication ability. All of these DIS-directed strategies rely on specifically targeting the viral RNA itself, which might not be practical given our inadequate knowledge of the overall structure of the HIV-1 5' region. The fact that RNA sequences such as SL1 and SL3 are known to form relevant RNA-protein interactions raises the possibility that the protein component of these interactions might also provide potential targets for anti-HIV therapy. Such approaches are currently being explored in research aimed at designing inhibitors of the TAR-Tat RNA-protein interaction [ 129 ]. Similar approaches might also be developed to target RNA-protein interactions involving SL1 or SL3 and Gag. Future directions Current HIV combination therapies have demonstrated that a multi-targeted approach against the virus results in the greatest degree of suppression of virus replication. Therefore, the identification of novel targets for anti-HIV therapy could significantly improve HIV treatment strategies. HIV-1 RNA dimerization is clearly a critical event that could be exploited as a target once its complete mechanism is elucidated. It is pleasing to see that a number of laboratories that have actively researched RNA dimerization and packaging are now moving beyond conventional in vitro and in vivo approaches toward more biologically relevant methods. One group has taken chemical modification protocols commonly used for in vitro RNA analysis, and adapted them for use in virus-producing cells. Hence, structural analysis of viral RNA, that would previously be carried out only in vitro on short fragments of artificially transcribed RNA, can now be performed on in vivo -generated HIV-1 genomic RNA (J.-C. Paillart and R. Marquet, personal communication, and [ 130 ]). This method also allows comparisons of cellular and virion-derived HIV-1 RNA and represents a middle ground between classic in vitro and in vivo approaches. The goal of this work is to provide insight on the true structure of the HIV-1 leader, and on which RNA substructures are involved in dimerization. Preliminary data suggest that viral RNA may already be dimerized in the cytoplasm (J-C. Paillart and R. Marquet, unpublished data). This method might also have application in regard to in vivo foot-printing that could allow the study of RNA-protein interactions in the context of virus-producing cells. The structure of the viral RNA that exists in the cell has long been a topic of interest, and recent data suggest that different RNA sequences might be involved in higher order intrastrand structures that favor the dimerization of the two RNA molecules. Such a model has been proposed [ 131 ], and is supported by numerous in vitro dimerization studies conducted on HIV-1, HIV-2, and SIV RNA [ 41 , 131 - 133 ]. The model proposes that the HIV-1 5' UTR can form two alternating conformations, termed the long-distance interaction (LDI) and the branched multiple hairpin (BMH) structures. The LDI conformation is believed to exist when the RNA is in a monomer form, and is thought to form a long extended base-paired structure with almost all of the proposed stem-loop sequences buried. This structure is thought to be favored during certain steps of the life cycle, such as translation. In this model, NC has been shown to bind the LDI structure to induce a switch to the BMH structure [ 131 ], in which the DIS and ψ would then be exposed in a manner able to mediate dimerization and packaging. Such a 'riboswitch' is an attractive hypothesis, especially since similar mechanisms have recently been proposed to account for previously unexplained results in the field of gene regulation [ 134 ]. Although there is currently little in vivo evidence directly supporting such a model in the case of retroviruses, the results of previous mutagenesis studies from several laboratories correlate with those that would be predicted from the riboswitch model, both concerning RNA packaging and RNA dimerization status [ 135 ]. In regard to dimerization being a prerequisite for packaging, it would also be interesting to test whether an HIV-1 RNA molecule in the LDI conformation can be packaged. Since the BMH conformation is believed to mediate dimerization, one would assume that the LDI structures would not be packageable if dimerization is truly a packaging prerequisite. Others have developed a fluorescence resonance energy transfer (FRET)-based system to allow visualization of RNA-Gag interactions within cells (A.M. Lever and co-workers, unpublished data). Such a system might provide insight into the timing of genome selection and packaging. It will also be interesting to determine whether this system can be adapted to pinpoint how retroviral RNA dimerization takes place within cells, and whether dimerization indeed occurs before RNA is selected for packaging. Competing interests None declared. Author's contributions RSR gathered the information discussed in this review, and was primary author of the manuscript. CL and MAW carefully read the manuscript and offered insightful suggestions for its revision. All authors read and approved the final version.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC516451.xml
543450	Effect of oligonucleotide primers in determining viral variability within hosts	Background Genetic variability in viral populations is usually estimated by means of polymerase chain reaction (PCR) based methods in which the relative abundance of each amplicon is assumed to be proportional to the frequency of the corresponding template in the initial sample. Although bias in template-to-product ratios has been described before, its relevance in describing viral genetic variability at the intrapatient level has not been fully assessed yet. Results To investigate the role of oligonucleotide design in estimating viral variability within hosts, genetic diversity in hepatitis C virus (HCV) populations from eight infected patients was characterised by two parallel PCR amplifications performed with two slightly different sets of primers, followed by cloning and sequencing (mean = 89 cloned sequences per patient). Population genetics analyses of viral populations recovered by pairs of amplifications revealed that in seven patients statistically significant differences were detected between populations sampled with different set of primers. Conclusions Genetic variability analyses demonstrates that PCR selection due to the choice of primers, differing in their degeneracy degree at some nucleotide positions, can eclipse totally or partially viral variants, hence yielding significant different estimates of viral variability within a single patient and therefore eventually producing quite different qualitative and quantitative descriptions of viral populations within each host.	Background One of the most difficult tasks faced by virologists is the documentation and evaluation of the genetic variability of viral populations in infected patients. These analyses are greatly facilitated by the use of the polymerase chain reaction (PCR). PCR based techniques do not always produce a highly specific and homogeneous product. When the template is a complex mixture of homologous sequences the aim of the amplification would be to preserve as much as possible the template-to-product ratios of every sequence in order to obtain a good representation of the diversity present in the initial sample. PCR products are derived from templates by a process involving complex chemical kinetics, and the relative abundance of the different homologous genomes among the final products is often a parameter of interest. This is the case, for instance, in experiments aimed at determining natural diversity in microbial communities [ 1 ] or at identifying members of multigene families [ 2 ] and it is of special relevance for studies of viral variability within hosts, especially for highly variable RNA viruses. The precise mechanisms involved in the preferential amplification of some templates from non-homogeneous sources are not fully understood and should be differentiated from those related to stochastic or tube-to-tube variations in amplification efficiency. When dealing with heterogeneous templates, two different processes can alter template-to-product ratios: PCR selection and PCR drift [ 3 ]. The former comprises mechanisms that favour the amplification of certain templates leading to their overrepresentation in the final product. Preferential denaturation due to GC content (in overall template and primer), differential efficiency of primer hybridisation or differential DNA polymerase extension rates (due to secondary structures of DNA) can all account for this type of bias. The second type of bias is related to stochastic variation in the early cycles of the reaction and its outcome should therefore be different in replicate PCR experiments. However, in a recent report analysing sampling strategies and repeatability to determine genetic variability in viral populations [ 4 ], we did not detect such PCR drift-caused bias. Given the high levels of variability found in RNA virus populations, primers involved in RT-PCR (retrotranscription followed by PCR) are usually designed as degenerate sequences to ensure that the chance of amplifying the different sequences present in an heterogeneous template will be more uniform and, therefore, all will be present in the amplified product in similar proportions to those in the original template. However, the use of even highly degenerate primers does not preclude the possibility of mismatches occurring between a given primer and some of the sequences present in a heterogeneous template, especially for highly variable regions. This would lead to differential amplification of sequences [ 5 ] (i.e. PCR selection, see above). Although unnoticed for the experimenter, if this preferential amplification does indeed occur, conclusions of many evolutionary studies, clinical predictions or even genotyping assessments would be affected. Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus of approximately 9.4 kb, classified in a separate genus (Hepacivirus) of the Flaviviridae family. HCV has been recognised as a major etiological agent of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma around the world [ 6 ]. HCV isolates can be highly divergent and have been classified into six major genotypes and more than 30 subtypes based on molecular phylogenetic analysis [ 7 ]. Moreover, like most RNA virus, HCV circulates in vivo as a highly polymorphic population of genetically closely related variants. This genetic variability may have implications not only for pathogenesis and prevention [ 8 ], but also for predicting the therapeutic outcome of HCV infection during interferon therapy [ 9 , 10 ]. Results Population and phylogenetic analyses Genetic variability in two different regions of the HCV genome was studied by means of RT-PCR amplification in eight infected patients. A fragment comprising partially E1 and E2 region including HVR1 and HVR2 (hypervariable regions 1 and 2, respectively) was amplified in six infected patients. In the other two patients, part of the NS5A region including the ISDR (interferon-sensitivity determining region) and the variable region 3 (V3) was amplified. Each fragment was amplified twice from each HCV infected patient with two slightly different sets of primers (Table 1 ). Differences between primer sets 1 and 2 for both regions are based on degeneracy of some nucleotide positions, with primer set 2 being more degenerate than primer set 1, except for primer 2-Ng2 (Table 1 ). After cloning and sequencing, about 100 sequences for the E1E2 region and about 50 sequences for the NS5A region were obtained from each patient and set of primers. Therefore, from each patient we obtained two different groups (populations) of sequences corresponding to the two parallel PCR reactions performed with primer sets 1 and 2 respectively. Table 1 List of primers for the E1E2 and NS5A regions of HCV Region Primer name a Nucleotide position b Primer set Sequence 5'-3' c Primer degeneracy E1E2 1-Eg1 1290–1309 1 CGCATGGC A TG G R A T ATGAT 2 2-Eg1 1290–1309 2 CGCATGGC Y T GG G A Y ATGAT 4 1-Eg2 1300–1321 1 GG R ATATGAT G ATGAA C TGGTC 2 2-Eg2 1300–1321 2 GG G ATATGAT R ATGAA Y TGGTC 4 1-Ea 1873–1854 1 GG A GTGAA G CA R TA T AC T GG 2 2-Ea 1873–1854 2 GG G GTGAA R CA R TA Y AC Y GG 16 NS5A 1-Ng1 6715–6739 1 TGGA Y GGGGTG C G C CT A CA T AGGT W 4 2-Ng1 6715–6739 2 TGGA C GGGGTG Y G M CT R CA Y AGGT T 16 1-Ng2 6734–6753 1 TAGGT W YGC S CCCCCYTGCA 16 2-Ng2 6734–6753 2 TAGGT T YGC G CCCCCYTGCA 4 1-Na 7519–7503 1 CC C TCSA GR GG G GGCAT 4 2-Na 7519–7503 2 CC Y TCSA RG GG R GGCAT 16 a g indicates genomic sense and a indicates antigenomic sense. b Nucleotide positions according to sequence accession no. M62321. c Nucleotides in bold indicate differences between primer sets and underlined nucleotides indicate overlapping positions of nested primers. Two types of population analysis were carried out ( Additional file 1 ) with the two groups of sequences from each patient: (a) common measures of genetic variability within each group of sequences, and (b) a population genetics test that detects differentiation between groups of sequences (permutation test of Hudson [ 19 ]). In addition, for each genomic region, a phylogenetic reconstruction using all detected haplotypes obtained with the two sets of primers was performed in order to visually inspect their distribution along the branches. Differences between groups of sequences from the same infected patient HCV genetic variability estimates from sequence data obtained for one of the two regions analysed for the eight infected patients showed a wide range of values ( Additional file 1 ). For the E1E2 region, the number of polymorphic sites ( S ) detected in a group of sequences from a particular set of primers ranged from 1 (in patient E04) to 68 (in patient E16). Similarly, for region NS5A, with only two patients analysed, S ranged from 4 to 70 for the same set of primers (patients N02 and N07, respectively). Haplotype diversity ( H T ) also reached both extreme values: in some groups of sequences corresponding to a particular primer set from a single patient (i. e. E10, E16, N07), almost every sequence constituted a different haplotype, resulting in H T ~ 1; whereas in other groups (i. e. E03, E04, E25) very few haplotypes were detected, with H T ~ 0.1 or even lower. When variability was measured taking both the frequencies of haplotypes and their genetic distances a wider variability range was observed: nucleotide diversity ( π ) of the viral populations estimated for the eight patients analysed differed by up to three orders of magnitude ( Additional file 1 ). The statistical significance of the observed differences in amount of genetic variation among groups of sequences could not be tested as no appropriate statistical test is currently available (but see [ 21 ]). However, the statistical significance of population differentiation can be evaluated between pairs of groups of sequences. In this way, for each patient the statistic and its corresponding P value was estimated using the permutation test [ 19 ]. Only for one patient the outcomes from the two parallel PCR amplifications were not significantly different. In patient E25, the use of different primers set seemed not to affect variability measures, although primer set 1 recovered more variability for all measurements than set 2. This is the only patient for which the average number of pairwise nucleotide substitutions ( k ) between both groups of sequences was lower than k obtained for a single primer set (e. g. primer set 2). Moreover, the main haplotype was similarly sampled by the two sets of primers (48 and 44 sequences respectively, see additional file 1 ), and therefore no significant statistical genetic differentiation between the two groups of sequences was detected. The distribution of viral sequences in the phylogenetic tree (Fig. 1a ) shows that sequence sampling with the two sets of primers can be considered very similar for this patient. Figure 1 Phylogenetic trees obtained by the neighbour-joining method for the different haplotypes of a) E1E2 region, patients E03, E04, E10, E16, E23 and E25; b) NS5A region, patients N02 and N07. Black dots represent haplotypes obtained with the set of primers 1; white dots represent haplotypes obtained with the set of primers 2, and grey dots are shared haplotypes obtained with both sets of primers. The number next to the dot indicates the number of times this haplotype was detected by a particular primer set. For shared haplotypes two numbers are given, the first one corresponds to primer set 1 and the second to primer set 2. The scale bar represents number of nucleotide substitutions per site (0.02 and 0.01, respectively). For the remaining patients we found that each set of primers produced a very different collection of viral sequences, leading to significant genetic differentiation between the two groups of sequences obtained with each primer set. However, in patients E10, E23 and N07, the total amount of variability recovered with both sets of primers was similar ( Additional file 1 ), although the distribution of haplotypes detected with each primer set in the phylogenetic tree was not homogeneous (Fig. 1a and 1b ). Moreover, population differentiation tests showed that the groups of sequences obtained with different primer sets for these three patients were significantly different. Even for patient E10, for which both groups of sequences were the most diverse of all analysed in this report and were apparently intermixed in the phylogenetic tree, the differentiation test detected significant differences. In patients E03, E04, E16 and N02 the population differentiation statistic ( ) also showed that the two groups of sequences obtained from each patient were statistically different, with genetic distances ( k ) between the two groups of sequences from the same patient largely exceeding the genetic distances estimated within a single group of sequences. Moreover, in these four patients, the amount of genetic variability detected also seemed to be deeply affected by the set of primers chosen for PCR (see additional file 1 ) with one set of primers recovering at least twice as many haplotypes as the other set. It is remarkable that in patients E03 and E04 the most frequently detected (main) haplotype with one set of primers was genetically distant from the main haplotype found with the alternative set, as shown by their relative positions in the phylogenetic tree (Fig. 1a ). It is also worth noticing that in patients E16 and E02 rare haplotypes were more abundant in PCR products obtained with primer set 2. Although we have dealt here with cloned sequences, direct sequencing of PCR products can also be much affected by PCR selection: both consensus sequences obtained from PCR amplification of the same cDNA aliquot from patient E04 with sets of primers 1 and 2 respectively, showed up to 7 nucleotide differences in 472 nucleotides (data not shown). Discussion PCR drift (i. e. bias in template-to-product ratios produced by random events occurred in the early cycles of the reaction) and PCR selection (i. e. differential amplification of specific sequences caused by differential annealing of oligonucleotide primers) are two processes that can lead to bias in template-to-product ratio of PCR amplifications [ 3 , 22 ]. In a previous report aimed at studying PCR repeatability on sampling HCV sequences from four infected patients [ 4 ] we found no evidence of such PCR drift under our conditions. However a role for PCR selection could not be ruled out. The main contributing factor to PCR selection is usually the differential affinity of primers for template sequences due to differences in the primary or secondary structure of DNA at target sites [ 3 ]. PCR assays rely on the efficient hybridization of primers to the target sequence. However, mismatches between the primer and the target molecules can affect duplex stability and may compromise the ability of the system to amplify and detect the target sequences. Numerous factors determine the final effect of mismatches, including primer length, nature and position of mismatches, hybridization temperature, presence of co-solvents (such as DMSO) and concentrations of both primers and monovalent and divalent cations. For instance, Ishii and Fukui [ 23 ] showed how using complex templates with different annealing temperatures severely affected the PCR outcome because of the presence of primer mismatch. Therefore, in samples with a heterogeneous composition of templates (such as viral populations in infected patients) the presence of mismatches can introduce differences in amplification efficiencies of the different templates hence leading to template-to-product ratios alterations during PCR. Some of these factors have been experimentally proven to cause this alteration. Attempts to reduce PCR bias caused by primer-template mismatches usually involve designing degenerate primers. Here we have studied the effect of small differences in primer degeneracy on PCR outcome with heterogeneous templates and their implications and extent on viral genetic variability at the intrapatient level. Our results indicate that template-to-product ratios can be significantly biased in standard PCR amplifications of non-homogeneous templates. By means of RT-PCR sampling of HCV sequences from eight infected patients we have found that sequence sampling from a single source varied considerably when two slightly different sets of primers were used in the PCR amplification: both sets of primers were chosen after inspection of HCV-1 aligned sequences from GenBank, and degenerations were introduced in both sets following only slightly different criteria. The use of one set of primers or another not only gave rise to different collections of sequences, but also to their different distribution along phylogenetic trees in most of viral populations studied. These results indicate that bias in template-to-product ratios can severely distort the results of analysis of variability in virus populations, both quantitatively (i. e. amount of genetic variability) and qualitatively (i. e. particular sequence clusters on a phylogenetic tree). Fan et al. [ 5 ] demonstrated that partially mismatched primers used in RT-PCR preferentially amplified different HVR1 sequences in a HCV virus population, but they pointed to the specific primer set used for the cDNA synthesis in the RT reaction as the main cause for the observed bias. To avoid this possible source of error, here we have used random hexamers to perform reverse transcriptions on viral RNA templates and consequently we have only focussed on the alterations caused by primers during PCR. This kind of PCR bias related to primer preferences to anneal to some viral templates has been previously demonstrated at the level of detection of HCV infections [ 24 ], detection of HCV mixed-genotype infections [ 25 ], differences in genotype assignment [ 26 ], or frequent failure to amplify hypervariable regions (references in [ 5 ]). But here we have demonstrated that this bias can be relevant at the population genetics level to the point that two independently obtained groups of sequences from the same patient could be considered as two significantly different viral populations. One consequence of our results is that viral variability estimated by means of PCR sampling of viral sequences will almost surely be an underestimation, and should always be considered as a minimum value. However relative changes in viral variability through time could probably be reliably assessed. Underestimates of variability due to PCR selection could be in agreement with the failure to find correlation between genetic diversity present in viral populations before treatment and treatment outcome [ 10 ], although significant changes in relative viral diversity yielded prognosis information. Another consequence is that, as recovered viral sequences after PCR are those more related to the sequences used in primer design, an unknown proportion of the original viral population present in the template source will not even be detected. This is shown in our experiment by the presence in the phylogenetic trees of divergent clusters of sequences from a single patient only amplified by one of the two sets of primers tested (see for instance patients E03, E04, E16, N02 and N07) in figure 1 . Since search for particular sequences related to therapy response is a common issue in antiviral resistance studies, this observation is of crucial interest as some sequences of specific or potential interest could remain unnoticed under particular PCR conditions. For example, in HCV the evolution of interferon sensitivity-determining region (ISDR) during IFN therapy is controversial [ 27 ]. It cannot be ruled out that discrepant results related to the predictive value of particular viral sequences detected by means of PCR could be partially due to the sampling bias of amplified sequences as those shown in the present report. The results obtained in this study allow us to strongly suggest that, for PCR-based variability studies, a certain level of primer degeneration, compatible with specific product amplification, would be more than advisable for variability studies in which, as with viral populations, there is no possibility of designing a perfect set of primer pairs that equally amplify all possible templates. For this, it is convenient to align many available related sequences and empirically determine which positions are most polymorphic and therefore susceptible to participate in mismatches. Conclusions PCR selection (differential amplification of specific sequences due to differential annealing of oligonucleotides) was detected and attributed to differences in degeneracy at some nucleotide positions in the oligonucleotides involved in amplification. Alterations in the template-to-product ratio during PCR amplification significantly affects viral population descriptions to the extent that two PCR outcomes from the same infected patient can result, after genetic population analyses, in two genetically distinct populations. Two important implications can be derived: first, all estimates of genetic variability parameters should be considered always as a minimum, and second, the search for particular existing genomes, such as drug resistant genomes, can be totally or partially eclipsed by others more susceptible to be annealed by the olinucleotides used in the amplification. Methods Viral RNA extraction and amplification Serum samples from eight patients infected with HCV were chosen for this study. Six patients infected with HCV-1b (E03, E04, E10, E16, E23 and E25) were analysed using two sets of primers that partially amplified the E1E2 region (472 nucleotides) and two patients infected with HCV-1a (N02 and N07) were analysed using two sets of primers that partially amplified the NS5A region (743 nucleotides). The first step in the design of oligonucleotide primers was to collect a representative variety of HCV-1 sequences from GenBank. From 50 homologous sequences, nucleotide positions for primers were chosen with GeneFisher [ 11 ], and two sets of homologous primers were designed for the E1E2 region and the same procedure for the NS5A region. For each region, both sets of homologous primers basically differ in their degree of degeneracy at some polymorphic positions (see table 1 ). Viral RNA was extracted from 140 μl of serum using High Pure Viral RNA Kit (Roche). In order to prevent any bias during reverse transcription reactions due to oligonucleotide specificity, all reverse transcription reactions were performed using random hexadeoxynucleotides. Reverse transcriptions (RT) were performed in a 20 μl volume containing 5 μl of eluted RNA, 4 μl of 5x RT buffer, 0.5 mM of each deoxynucleotide, 0.5 μg of random hexamers, 100 U of MMLV reverse transcriptase (Promega), and 20 U of RNasin Ribonuclease Inhibitor (Promega). Reactions were incubated at 37°C for 60 min, followed by 2 min at 95°C. A first PCR round was then carried out in a 100 μl volume containing 10 μl of the reverse transcription product, 0.2 mM of each dNTP, 400 nM of genomic primer and 400 nM of antigenomic primer and 1.25 units of Pfu DNA polymerase (Promega). The outer set of primers for the E1E2 region were 1-Eg1 (or alternatively 2-Eg1) and 1-Ea (or 2-Ea) (see Table 1 ). Hemi-nested PCR was carried out to amplify a 472-bp fragment with nested primer 1-Eg2 (or 2-Eg2) and original primer 1-Ea (or 2-Ea). The outer set of primers for the NS5A region were 1-Ng1 (or 2-Ng1) and 1-Na (or 2-Na). Hemi-nested PCR were carried out to amplify a 743-bp fragment with 1-Ng2 (or 2-Ng2) and 1-Na (or 2-Na). All reactions were performed in a Perkin Elmer 2400 thermalcycler according to the following profile: initial denaturation at 94°C for 1 min; 5 cycles at 94°C for 30 s, 55°C 30 s, 72°C 3 min; then 35 cycles at 94°C 30 s, 52°C 30 s, 72°C 3 min, and a final extension at 72°C for 10 min. A single amplified product was observed after electrophoresis on 1.4 % agarose gels stained with ethidium bromide. The same PCR conditions were strictly applied to every primer set in both regions. The 233 newly reported sequences (haplotypes) are deposited in the EMBL nucleotide sequence database under accession numbers AF715552-AF715784. Cloning and sequencing of viral populations Amplified products from the second round of PCR for the E1E2 and NS5A regions were purified using High Pure PCR product Purification Kit (Roche) and directly cloned into EcoRV-digested pBluescript II SK (+) phagemid (Stratagene). Recombinant plasmid DNA was purified using the High Pure Plasmid Isolation Kit (Roche). Cloned products were sequenced using vector-based primers KS and SK (Stratagene). Sequencing was carried out using ABI PRISM BigDye Terminator v3.0 Ready Reaction Cycle Sequencing KIT (Applied Biosystems) on an ABI 3700 automated sequencer. Sequences were verified and both strands assembled using the Staden package [ 12 ]. Phylogenetic reconstruction and population genetics analysis Sequences were aligned using CLUSTALX v1.81 [ 13 ]. The neighbour-joining algorithm [ 14 ] applied on the pairwise nucleotide divergence matrix using Kimura's two parameter model [ 15 ] was used to obtain phylogenetic trees using the MEGA program [ 16 ]. Polymorphism and genetic differentiation were analysed using DNAsp version 4.0 [ 17 ]. Estimated polymorphism parameters included: number of polymorphic sites ( S ); haplotype diversity ( H T ) considering as haplotype each different sequence; nucleotide diversity ( π ) [ 18 ]; and average number of pairwise differences between sequences ( k ). Genetic differentiation between groups of sequences was estimated as the average number of nucleotide substitutions between groups ( d xy ). The statistical significance of genetic differentiation between groups, as estimated by , was established by the permutation test [ 19 ]. The proportion of nucleotide diversity attributable to variation between populations, the fixation index F st , was calculated using the ARLEQUIN program ver. 2.000 [ 20 ]. Competing interests The authors declare that they have no competing interests. Authors' contributions MAB and IG-R co-conceived, designed and coordinated the study, participated in the molecular studies and sequence alignment, interpreted data, oversaw the training of technicians, and co-drafted the manuscript; MAB isolated viral genomes, co-performed population and phylogenetic analyses; NJ and MT-P participated in molecular studies and sequence alignment, interpreted the data and helped draft the manuscript; AM interpreted data and participated in proofreading of the manuscript; FG-C coordinated the study, interpreted data, co-performed population and phylogenetic analyses and participated in proofreading of the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional File 1 Summary of genetic variability and population differentiation of within patient HCV populations based on viral sequences obtained with two alternative primer set. E1E2 region was analysed in patients E03, E04, E10, E16, E23 and E25, and NS5A region in patients N25 and E02 Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC543450.xml
529306	Influence of oxygen tension on myocardial performance. Evaluation by tissue Doppler imaging	Background Low O 2 tension dilates coronary arteries and high O 2 tension is a coronary vasoconstrictor but reports on O 2 -dependent effects on ventricular performance diverge. Yet oxygen supplementation remains first line treatment in cardiovascular disease. We hypothesized that hypoxia improves and hyperoxia worsens myocardial performance. Methods Seven male volunteers (mean age 38 ± 3 years) were examined with echocardiography at respiratory equilibrium during: 1) normoxia (≈21% O 2 , 79% N 2 ), 2) while inhaling a hypoxic gas mixture (≈11% O 2 , 89% N 2 ), and 3) while inhaling 100% O 2 . Tissue Doppler recordings were acquired in the apical 4-chamber, 2-chamber, and long-axis views. Strain rate and tissue tracking displacement analyses were carried out in each segment of the 16-segment left ventricular model and in the basal, middle and apical portions of the right ventricle. Results Heart rate increased with hypoxia (68 ± 4 bpm at normoxia vs. 79 ± 5 bpm, P < 0.001) and decreased with hyperoxia (59 ± 5 bpm, P < 0.001 vs. normoxia). Hypoxia increased strain rate in four left ventricular segments and global systolic contraction amplitude was increased (normoxia: 9.76 ± 0.41 vs hypoxia: 10.87 ± 0.42, P < 0.001). Tissue tracking displacement was reduced in the right ventricular segments and tricuspid regurgitation increased with hypoxia (7.5 ± 1.9 mmHg vs. 33.5 ± 1.8 mmHg, P < 0.001). The TEI index and E/E' did not change with hypoxia. Hyperoxia reduced strain rate in 10 left ventricular segments, global systolic contraction amplitude was decreased (8.83 ± 0.38, P < 0.001 vs. normoxia) while right ventricular function was unchanged. The spectral and tissue Doppler TEI indexes were significantly increased but E/E' did not change with hyperoxia. Conclusion Hypoxia improves and hyperoxia worsens systolic myocardial performance in healthy male volunteers. Tissue Doppler measures of diastolic function are unaffected by hypoxia/hyperoxia which support that the changes in myocardial performance are secondary to changes in vascular tone. It remains to be settled whether oxygen therapy to patients with heart disease is a consistent rational treatment.	Introduction Low oxygen tension dilates coronary arteries and high oxygen tension is a coronary vasoconstrictor. Yet oxygen supplementation remains first line treatment in cardiovascular disease states such as myocardial infarction and pulmonary oedema. Endothelium-dependent vasodilation is reduced in patients with ischemic heart disease [ 1 ] and such patients are believed to have a reduced ability to counteract the circulatory consequences of systemic (air line travel, high altitude stay) and regional (coronary artery stenosis) hypoxia. Spontaneous nocturnal hypoxia with desaturation for hours is a frequent phenomenon in patients with severe coronary artery disease [ 2 ]. Left atrial, left ventricular (LV), and right ventricular (RV) end-systolic diameter fall during simulated extreme altitude [ 3 ] and during moderate altitude exposure [ 4 ]. There is controversy concerning myocardial performance during hypoxia; improvement [ 4 ], no change [ 3 , 5 , 6 ]. and worsening [ 7 ] of left ventricular systolic function, have been described. One study looked at diastolic function expressed as E/A ratio and found a reduction with hypoxia [ 3 ]. Hyperoxia, a condition in which the total oxygen content of the body is increased above that normally existing at sea level, is associated with impairment of cardiac relaxation and increased left ventricular filling pressures in patients with and without congestive heart failure [ 8 ]. Tissue Doppler imaging (TDI) objectively derives measurements of contraction and relaxation velocities directly from the myocardium and thus yields information not previously accessible by echocardiography [ 9 ]. We used TDI to study myocardial performance in healthy volunteers and we hypothesized that hypoxia increases and hyperoxia reduces myocardial performance. Material and Methods Subjects Seven healthy men, (age 25–46 (mean 38) years) completed the study. All subjects were normotensive, non-smokers, on no medication, had a normal left ventricular function by 2-D echocardiography, and had no family history of ischemic heart disease. The local ethical committee approved the study. Ventilation system We used a system consisting of a ventilator, a gas analyser with pulse oximeter, and a computer. Computer programs control the experimental procedure and continuously collect data from the ventilator and gas analyser [ 10 ]. Echocardiography Echocardiography was performed from the apical acoustic window. Tissue Doppler recordings were acquired as digital loops in the apical 4-chamber, 2-chamber, and long-axis views [ 9 ]. To avoid aliasing, the settings of the ultrasound equipment and colour-coded area were adjusted to obtain the highest possible frame rate. We measured peak strain in the 16-segment left ventricular model and in 3 segments in the right. From the color-coded tissue tracking image, the motion amplitude toward the apex in systole was recorded in each segment. The global systolic contraction amplitude (GSCA) was calculated as the average shortening amplitude of all 16 segments. The peak E velocity was obtained by pulsed Doppler measurements of the mitral inflow at the tip of the mitral leaflets. We further assessed isovolumetric acceleration, TEI index (spectral and TDI), tricuspid regurgitation and pulmonary ejection time. The tissue E' velocity was obtained by Tissue Doppler at the lateral mitral annulus. E/E' has been proposed as a tool for assessing LV filling pressures that combines the influence of transmitral driving pressure and myocardial relaxation [ 11 ]. The TEI index (spectral or TDI), a combined measure of systolic and diastolic function, was assessed by Doppler time intervals from the mitral inflow and LV outflow tract or the time intervals were obtained by TDI at the lateral mitral annulus. The TEI index was calculated by a-b/b where the time interval "a" was measured from cessation to onset of mitral inflow and the time interval "b" was the duration of the LV outflow velocity profile. To minimize the variability of the measurements, all ECHO recordings were performed and analyzed in a blinded fashion by the same author (P.S.). Study protocol TDI was performed when the subjects had rested for 15 minutes breathing room air (SpO 2 97.9 ± 0.1 %), after 5 minutes of respiratory equilibrium during hypoxia (SpO 2 77.6 ± 1.2 %), and after 5 minutes of respiratory equilibrium during hyperoxia (SpO 2 99.0 ± 0.2 %). Heart rate was continuously recorded on computer by means of the pulse oximeter. Blood pressure was measured once during each of the three respiratory steady state situations by an automatic blood pressure measuring device based on the oscillometric method. Statistical analysis All data are presented as mean ± SEM. Comparisons of the responses to changes in F i O 2 (normoxia, hypoxia and hyperoxia) were made with a one-way repeated-measures analysis of variance. The Student-Newman-Keuls test was used post hoc to identify pairwise differences. Differences were considered statistically significant when P < 0.05. Results Respiratory parameters and hemodynamics FiO 2 and FeO 2 decreased with hypoxia and increased with 100% oxygen breathing (table 1). FeCO 2 and tidal volume were unchanged in all three test situations, reflecting that the subjects were in respiratory steady state. There was a small, but statistically significant decrease in respiratory rate from normoxia to hypoxia, which might reflect that the subjects were more accustomed to the test situation at this stage. There was a significant increase in heart rate with hypoxia and a decrease with hyperoxia (figure 1 ). Neither systolic (126 ± 6, 123 ± 8, 122 ± 12 mmHg, respectively, P = ns) nor diastolic blood pressure (80 ± 5, 78 ± 8, 81 ± 6 mmHg, respectively, P = ns) changed significantly with test situation. Figure 1 Bar graph depicting heart rate during normoxia, hypoxia and hyperoxia. ** P < 0.01 vs. normoxia. TDI and spectral echocardiography, hypoxia A significant increase in strain rate was found in 4 segments (figure 2 ). GSCA increased with hypoxia (9.76 ± 0.41 vs. 10.87 ± 0.42, P < 0.001, figure 3 ). Tissue tracking displacement was reduced in all three right ventricular segments (figure 4 ) and systolic tricuspid regurgitation increased with hypoxia (figure 5 ). The TEI index (spectral or TDI) and E/E' did not change with hypoxia. Figure 2 Comparison of peak systolic strain in the 16-segment left ventricular model illustrating the effects of hypoxia and hyperoxia. A , Anterior; B , basal; D , distal; I , inferior; L , lateral; M , mid; P , posterior; S , septal. * P < 0.05, ** P < 0.01 vs. normoxia. Figure 3 Tissue tracking score index on the basis of the 16-segment left ventricular model illustrating the effects of hypoxia and hyperoxia. Figure 4 Comparison of peak systolic strain in the right ventricle model illustrating the effects of hypoxia and hyperoxia. * P < 0.05. Figure 5 Bar graph depicting systolic tricuspid regurgitation during normoxia, hypoxia and hyperoxia. ** P < 0.01 vs. normoxia. TDI and spectral echocardiography, hyperoxia Hyperoxia worsened left ventricular function. Strain rate was reduced in 10 segments (figure 2 ) with preponderance in the lateral and anterior segments. GSCA was reduced (8.83 ± 0.38, P < 0.001 vs. normoxia). Tissue tracking displacement did not change in the right ventricular segments (figure 4 ) and systolic tricuspid regurgitation was unchanged compared with normoxia (figure 5 )). The TDI TEI index was significantly increased with hyperoxia (0.32 ± 0.04 vs. 0.45 ± 0.05, P < 0.001) and the spectral TEI index showed similar changes. E/E' did not change with hyperoxia. Discussion The main findings of the present study of healthy volunteers are: 1) hypoxia increased strain rate and tissue tracking displacement. 2) Hypoxia increased tricuspid regurgitation and reduced right ventricular tissue tracking displacement. 3) Hyperoxia reduced strain rate and tissue tracking displacement and increased the TEI index. The novelty of our findings, when compared to the literature, is the demonstration that longitudinal myocardial function, and thus the function of the subendocardium, is sensitive to moderate changes in inspired oxygen. Patients with ischemic heart disease and heart failure frequently encounter hypoxia but the consequences of hypoxia on left ventricular function remain a matter of controversy, in part because of differences in methodology and the measured parameters. In early studies using roentgenkymograms [ 12 ] and dye injection [ 13 ] hypoxia was shown to increase cardiac output at rest despite reduced [ 13 ] or unchanged [ 12 ] stroke volume. The authors explained the increase in cardiac output by increased heart rate [ 12 , 13 ] Myocardial blood flow in the left and right ventricles increased at high altitude in dogs studied with radioactive microspheres [ 14 ] and in healthy controls using positron emission tomography [ 15 ]. In a study using M-mode echocardiography at high altitude [ 4 ] percent fractional shortening and velocity of circumferential fiber shortening remained normal while LV isovolumetric contraction time shortened. Echocardiography was also employed in a simulated ascent of Mount Everest [ 3 ] and an insignificant increase in fractional shortening and ejection fraction was found. It is generally accepted that right-sided pressures increase with hypoxia [ 3 , 14 ] We speculate that the improvement in tissue tracking displacement in our study reflects the systemic vasodilation which is another consequence of hypoxia [ 16 ]. On the other hand, RV-systolic function decreased which is likely to be correlated to the increased systolic pulmonary pressure reflecting the increased pulmonary vascular resistance during hypoxia. Hemoglobin saturation in healthy persons increase very little from breathing room air to breathing 100% oxygen. Nevertheless, profound cardiovascular effects were found. This is probably because of an anticipated increase in oxygen dissolved into plasma from 0.32% to 2.09% [ 17 ]. Hyperoxia is a possible product of oxygen therapy when administered to patients with heart disease during acute illness. Hyperoxia reduces cardiac output as documented with roentgenkymograms [ 12 ], dye injection [ 18 , 19 ]., echocardiography [ 20 ] heart catheterisation [ 8 ] and indirectly by measurement of isometric systolic tension by means of strain gauge in dogs [ 21 ]. Reduced cardiac output with hyperoxia has even been demonstrated in patients with myocardial infarction using dye injection [ 22 ]. Some of the reduction in cardiac output may be explained by the observation that hyperoxia reduces heart rate as seen in our study and previously [ 19 , 23 - 25 ] Reduced heart rate is, however, not an entirely consistent finding [ 8 , 20 , 22 , 26 ] but in these four studies this could be because of high sympathetic tone (stay in a hyperbaric chamber [ 20 ], acute myocardial infarction [ 22 ], open heart surgery [ 26 ], heart catheterisation [ 8 ]). We found that heart rate increased with hypoxia and decreased with hyperoxia. This might have affected our measures of LV systolic and diastolic function but we consider this unlikely on the basis of our previous studies [ 11 , 27 ] demonstrating that TEI index, strain rate and the tissue tracking was unrelated to heart rate. During hypoxia as well as hyperoxia no change in LV filling pressure was noted as the E/E' ratio was unchanged. The individual parameters in the E/E' ratio are known to be influenced by heart rate but as a ratio it seems independent of heart rate and load conditions [ 11 , 27 ]. Therefore, we did not perform any adjustments for heart rate in the evaluation of LV diastolic function. Because of the finding of no change [ 19 ] or a discrete rise [ 18 , 22 ] in blood pressure, a reduction in cardiac output results in an increased systemic vascular resistance during hyperoxia [ 18 , 19 , 22 ] Regardless of the fact that sympathetic tone may be affected by hyperoxia, even after complete sympathetic blockade myocardial contractile force remains reduced [ 26 ]. In the present study both tissue tracking displacement and strain rate worsened during hyperoxia. It seems plausible that this deterioration could be explained by systemic vasoconstriction [ 21 ] increasing afterload. In conclusion, hypoxia improves and hyperoxia worsens systolic myocardial performance in healthy male volunteers. TDI measures of diastolic function are unaffected by hypoxia/hyperoxia which support that the changes in myocardial performance are secondary to changes in vascular tone. It remains to be settled whether oxygen therapy to patients with heart disease is a rational treatment that may sometimes be harmful or whether supplemental oxygen consistently results in an overall gain in delivered oxygen.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC529306.xml
555959	Placentation in the paca (Agouti paca L)	Background The paca is a South American rodent with potential as a commercial food animal. We examined paca placenta as part of a wider effort to understand the reproductive biology of this species. Methods Thirteen specimens between midgestation and term of pregnancy were studied by light and transmission electron microscopy. Results The placenta is divided into several lobes separated by interlobular trophoblast. Maternal arterial channels and fetal veins are found at the centre of each lobe. In the labyrinth, maternal blood flows through trophoblast-lined lacunae in close proximity to the fetal capillaries. The interhaemal barrier is of the haemomonochorial type with a single layer of syncytiotrophoblast. Caveolae occur in the apical membrane of the syncytiotrophoblast and recesses in the basal membrane, but there is no evidence of transtrophoblastic channels. The interlobular areas consist of cords of syncytiotrophoblast defining maternal blood channels that drain the labyrinth. Yolk sac endoderm covers much of the fetal surface of the placenta. The subplacenta comprises cytotrophoblast and syncytiotrophoblast. There are dilated intercellular spaces between the cytotrophoblasts and lacunae lined by syncytiotrophoblast. In the junctional zone between subplacenta and decidua, there are nests of multinucleated giant cells with vacuolated cytoplasm. The entire placenta rests on a pedicle of maternal tissue. An inverted yolk sac placenta is also present. The presence of small vesicles and tubules in the apical membrane of the yolk sac endoderm and larger vesicles in the supranuclear region suggest that the yolk sac placenta participates in maternal-fetal transfer of protein. Conclusion The paca placenta closely resembles that of other hystricomorph rodents. The lobulated structure allows for a larger exchange area and the development of precocial young.	Introduction The paca ( Agouti paca , L) is a South American rodent that lives in forested habitats near water and feeds largely on fallen fruit. It is hunted for its meat, which is considered a delicacy, and is an important source of animal protein for rural populations. This has led to indiscriminate exploitation, resulting in a significant reduction in the population density of this species in Brazil. We here describe the morphology of paca placenta as revealed by light microscopy and transmission electron microscopy. This study is part of a wider effort to document the reproductive physiology of paca. It is hoped that the information obtained will contribute to a rational strategy for conservation of the species and possibly for production, as paca has great potential as a commercial food animal [ 1 ]. Materials and methods The observations are based on placentae collected from 13 pregnant females. One was in early gestation, two in midgestation and nine near term of pregnancy. This material was collected at Paulista State University, Jaboticabal, Sao Paulo, Brazil. The research was authorized by the Brazilian Institute of the Environment and Renewable Natural Resources (IBAMA). The experimental protocol was approved by the Bioethics Committee of the School of Veterinary Medicine, University of Sao Paulo. The animals were sedated with azaperone (Stresnil, Janssen Pharmaceutica, Brazil; 0.1 mg/kg I.M.) and given atropine (0.5 mg I.M.). Anaesthesia was induced with xylazine (Coopazine, Coopers Brazil, Sao Paulo, S.P., Brazil; 1.5 mg/kg I.M.) and ketamine (Holliday Scott S.A., Brazil; 20 mg/kg I.M.). Hemihysterectomy was then performed under aseptic conditions during inhalation anaesthesia with halothane (Hoechst, Frankfurt, Germany). Postoperatively the animals were treated with benzyl penicillin and streptomycin (Pentabiotico ® , Fort Dodge, Campinas, S.P., Brazil; 8,000–24,000 IU/kg I.M.) and an analgetic (buprenorphine, Temgesic ® , Schering-Plough, S.P., Brazil). A detailed description of the anaesthesia and surgical procedures has been published elsewhere [ 2 ]. Pieces of ten placentae were fixed in Bouin's solution or 10% formaldehyde and processed by standard histological procedures for embedding in paraplast, then sectioned at 7 μm (automatic microtome, Model RM2155, Leica, Germany). Sections were stained with haematoxylin and eosin, Masson's trichrome or Gomori's trichrome or by the periodic acid Schiff (PAS) reaction with haematoxylin as counterstain. Seven placentae were processed for transmission electron microscopy. Small samples were fixed in 2.5% glutaraldehyede in 0.1 M phosphate buffer, pH 7.4, washed in buffer and post-fixed in 1% osmium tetroxide (Polysciences, Warrington, PA, USA). They were then dehydrated, washed with propylene oxide and embedded in Spurr's resin (Spurr's Kit, Electron Microscopy Sciences, CO, U.S.A.). 60 nm sections were made and stained with 2% uranyl acetate (5 minutes) and 0.5% lead citrate (10 minutes). The ultrastructural observations were made with a transmission electron microscope (JEOL 1010, Peabody, MA, U.S.A). Results The overall plan of the paca placenta is shown schematically in Figure 1 . The labyrinth is divided into lobes separated by interlobular trophoblast. Beneath this is the subplacenta, a structure unique to hystricomorph rodents. The junctional zone between these structures and the maternal decidua contains nests of giant cells. The placenta is attached to the uterus by a pedicle of maternal tissue. In addition, there is an inverted yolk sac placenta, which connects with the fetal surface of the chorioallantoic placenta. Figure 1 Schematic drawing of the paca placenta. The labyrinth is divided into lobes separated by interlobular trophoblast. Beneath it is found the subplacenta and then the decidua. There is a tenuous attachment to the uterine wall, the pedicle or mesoplacenta. An inverted yolk sac placenta is present throughout gestation. Histology The centres of the lobes, the labyrinth and the interlobular regions are clearly defined (Figure 2A ). The central region of each lobe contains fetal and maternal vessels around which there is a considerable quantity of connective tissue (fetal mesenchyme; Figure 2B ). The vessels carrying maternal arterial blood lack endothelium and they are lined by trophoblast cells (Figure 2C ). Figure 2 The labyrinth of the paca placenta. (A) The central region of a lobe (cl), a labyrinthine region (lab) and interlobular regions (in). Haematoxylin and eosin. (B) Central region of a placental lobe, showing the presence of fetal veins (fv), maternal arterial blood spaces (ma) lined by trophoblast cells, and the mesenchyme (mes) surrounding these structures. Haematoxylin and eosin. (C) Detail to show the trophoblastic lining of maternal blood spaces (tr) and the intact walls of small fetal veins. PAS. (D) The placental labyrinth, showing the radial disposition (←) of the trophoblastic columns, the fetal capillaries (fc) and maternal blood spaces (mbs). Haematoxylin and eosin. Scale bars: 500 μm (A), 200 μm (B); 50 μm (C, D). The most extensive portion of the lobe is the labyrinth. Due to close proximity between maternal and fetal blood vessels, it is the region where most maternal-fetal exchange takes place. The maternal blood spaces or lacunae are not lined by endothelium; they are defined by trophoblastic cell columns or cords. The cell columns are radially arranged as is apparent when the lobe is seen in cross section (Figure 2D ). The syncytial nature of the trophoblastic columns is indicated by the close proximity of their nuclei. The interlobular regions comprise cords of syncytiotrophoblast with abundant basophilic cytoplasm. These cords define maternal blood channels. The channels converge upon larger blood spaces, still without an endothelial lining, that receive blood from two or more adjacent lobes. Thus, each interlobular region is common to several lobes (Figure 3A ). The interlobular regions also contain fetal arteries (Figure 3B ) that give rise to the capillaries of the labyrinth. Figure 3 The interlobium of the paca placenta. (A) Channels in the interlobium (in) drain the maternal blood spaces of the labyrinth (lab) and converge on larger venous blood spaces (mv). Haematoxylin and eosin. (B) Fetal artery (fa) at the border between an interlobular region and the labyrinth at the periphery of a lobe. Haematoxylin and eosin. Scale bars: 100 μm (A); 50 μm (B). Placental surface Most of the surface of the placental disk is covered by an epithelium formed by the endoderm of the parietal yolk sac (Figure 4A–C ). An almost continuous Reichert's membrane can be demonstrated (Figure 4B ). Beneath it is a layer of spongiotrophoblast. These cells differ from those that occur in the marginal and interlobular trophoblast. They are larger in size and have a rounded and vacuolated appearance (Figure 4C ). The centre of the fetal surface of the placenta, including the attachment of the cord, is covered by a layer of connective tissue (Figure 4D ). Figure 4 Fetal surface of the paca placenta. (A) The visceral yolk sac (vys) attaches to the surface of the chorioallantoic placenta. Just before attachment it forms the fibrovascular ring (fvr). Left of the point of attachment, the endoderm continues as the parietal yolk sac (pys). To the right, the surface is covered by connective tissue. Masson's trichrome. (B) The parietal yolk sac endoderm (endo) forms a layer of epithelial cells that rests on Reichert's membrane (Rm). Immediately below the membrane are scattered connective tissue cells (ct). Masson's trichrome. (C) Beneath the endoderm and Reichert's membrane are spongiotrophoblast cells (sp tr), with a vacuolated appearance, marginal syncytium (ma tr) and a portion of the labyrinth (lab). Haematoxylin and eosin. (D) The centre of the placental disk is covered by connective tissue. Beneath this are marginal trophoblast and labyrinth. Haematoxylin and eosin. Scale bars: 500 μm (A); 10 μm (B); 100 μm (C-D). Subplacenta, junctional zone and pedicle The subplacenta is organized as folded lamellae of cytotrophoblasts supported on a thin layer of mesenchyme carrying fetal vessels (Figure 5A ). The cytotrophoblast is multilaminar and mitotic figures are common here in early and midgestation. Beneath this layer is syncytiotrophoblast, which contains vacuoles and PAS-positive material. In late gestation this region becomes more compact and the entire subplacenta undergoes a process of degeneration. Figure 5 Subplacenta, junctional zone and pedicle of the paca placenta. (A) Subplacenta. A layer of cytotrophoblasts (cy tr) is supported on lamellae of fetal mesenchyme (fm). Beneath it is the subplacental syncytiotrophoblast (sy tr). Gomori's trichrome. (B) Groups of multinucleated giant cells with a finely granular cytoplasm are found between the subplacenta and decidua. They are bordered by connective tissue. Masson's trichrome. (C) Middle portion of the placental pedicle, showing a large number of maternal blood vessels (mbv) and connective tissue fibers (ct). Haematoxylin and eosin. Scale bars: 20 μm (A-B); 200 μm (C). Groups of giant cells are found in the junctional zone between the subplacenta and decidua (Figure 5B ). They are multinucleated and have a finely granular, basophilic cytoplasm. They are PAS-positive. The placenta is attached to the uterus by a placental pedicle made up of uterine tissue. In the upper region of this pedicle, a fine and discontinuous layer of connective tissue is interposed between the maternal tissue and fetal trophoblast. In the middle portion of the pedicle, a large number of vessels pass to or from the placenta. This region is characterized by dense fibres of connective tissue externally and of looser connective tissue around the vessels (Figure 5C ). A layer of squamous epithelial cells with simple, round nuclei, lightly condensed chromatin and clear cytoplasm covers the entire pedicle (not shown). Yolk Sac Placenta There is an inverted yolk sac placenta, which is attached to the fetal surface of the chorioallantoic placenta. Just before attachment it forms the fibrovascular ring (Figure 4A ). The yolk sac exhibits numerous digitiform projections (Figure 6A ), which sometimes are branched. They consist of a mesenchymal core covered by a layer of endoderm. The latter forms a simple columnar epithelium of cells with apically situated cell nuclei (Figure 6B ). Figure 6 Yolk sac placenta of the paca. (A) The visceral yolk sac is complexly folded with columnar epithelial cells of endodermal origin (endo) supported by fetal mesenchyme (fm). Haematoxylin and eosin. (B) The mesenchyme (stained blue) contains vitelline blood vessels (vit bv). Masson's trichrome. Scale bars: 50 μm (A); 10 μm (B). Ultrastructure The paca placenta is of the syncytial haemomonochorial type. A single trophoblast layer can be identified between the blood in the maternal blood spaces of the labyrinth and the endothelium of the fetal capillaries (Figure 7A–B ). This trophoblast is syncytial in nature without cell boundaries and with large nuclei, often in close proximity to one other. Microvilli project from the apical membrane into the maternal blood space. Caveolae are seen in the apical membrane and the syncytiotrophoblast contains coated vesicles and larger vacuoles. There are recesses in the basal membrane. However, we saw no evidence of transtrophoblastic channels. There is an abundant amount of rough endoplasmic reticulum, a Golgi apparatus, lysosomes and numerous mitochondria. Figure 7 Ultrastructure of the labyrinth, interlobium and parietal yolk sac endoderm. (A-B) The interhaemal barrier. Only a single layer of syncytiotrophoblast is interposed between the blood flowing in the maternal blood spaces (mbs) and the endothelium of the fetal capillary (fc). The apical membrane contains invaginations (arrowheads) and the cytoplasm includes small coated vesicles (cv) and larger vacuoles. There are recesses in the basal membrane (arrows). (C) Interlobium. Most of the trophoblast is syncytial (sy tr) with abundant rough endoplasmatic reticulum. There are many microvilli where it is in contact with the maternal blood space. Cytotrophoblast cells with large nuclei are found in some regions of the interlobium. Desmosomes are found between the lateral membranes of adjacent cells (arrowheads). (D) Parietal yolk sac endoderm. These columnar cells have numerous microvilli at the apical surface. The supranuclear cytoplasm contains vacuoles and vesicles, tubular mitochondria and rough endoplasmic reticulum. Desmosomes are found between the lateral membranes of adjacent cells (arrowheads). Scale bars: 1 μm (A-B); 5 μm (C); 2 μm (D). In the interlobular areas, the syncytiotrophoblast bordering the maternal blood spaces has numerous microvilli (Figure 7C ). The cytoplasm has abundant rough endoplasmic reticulum, mitochondria and electron-dense droplets. Cytotrophoblast cells occur within the syncytium. Desmosomes are present between adjacent cytotrophoblast cells as well as between cytotrophoblasts and the overlying syncytium. The cells of the parietal yolk sac endoderm form a columnar epithelium (Figure 7D ). These cells are irregular in shape with basally situated nuclei. The apical membrane, which faces the uterine lumen, has microvilli and caveolae. The supranuclear cytoplasm contains vacuoles and vesicles, tubular mitochondria and rough endoplasmic reticulum. Desmosomes are found between the lateral membranes of adjacent cells. Subplacenta and junctional zone The cytotrophoblast layer is multilaminar with dilated intercellular spaces (Figure 8A ). The cytoplasm of the syncytium has mitochondria, rough endoplasmic reticulum, numerous electron-dense granules (Figure 8B ) and large accumulations of electron-dense material. The cytoplasm has electron transparent patches, which gives it a vacuolated appearance. There are lacunae within the syncytiotrophoblast, lined by microvilli and containing material of moderate electron density (Figure 8B ). Desmosomes occur between adjacent cytotrophoblast cells and between the plasma membranes of these cells and that of the syncytial trophoblast. Figure 8 Ultrastructure of the subplacenta and junctional zone of the paca placenta. (A) The cytotrophoblast layer (cy tr) is multilaminar with dilated intercellular spaces. The cells are characterized by their large nuclei. They rest on a thin basement membrane (bm) which separates them from the fetal mesenchyme. (B) The syncytiotrophoblast (sy tr) contains electron-dense droplets (dd). Microvilli project from the syncytiotrophoblast into lacunae containing material of moderate electron density (arrows). (C) Multinucleated giant cells from the junctional zone. The cytoplasm has electron transparent areas, giving it a vacuolated appearance. (D) Two giant cells (gc) separated by intercellular matrix into which they send processes (arrows). Scale bars: 5 μm (A-C); 1 μm (D). In the junctional zone between the decidua and the lateral aspect of the subplacenta, there are nests of multinucleated giant cells (Figure 8C ). Their morphology is variable. The cytoplasm has extensive electron transparent areas. The organelles tend to be confined to the perinuclear and marginal areas (Figure 8D ) and include mitochondria, rough endoplasmic reticulum and electron-dense granules. The giant cells are separated by electron-dense material into which they send processes (Figure 8D ). Yolk sac placenta The apical surface of the endoderm cells has numerous microvilli of relatively uniform length (Figure 9A ). Small vesicles and tubules are present in the most apical regions of the cytoplasm (Figure 9B ). The supranuclear cytoplasm contains a number of larger vesicles and vacuoles with a variable amount of electron-dense content. The perinuclear cytoplasm also houses a small Golgi complex. The cytoplasm has mitochondria and rough endoplasmic reticulum. Desmosomes and terminal bars are present between the lateral membranes of the cells. Figure 9 Ultrastructure of the inverted yolk sac placenta of the paca. (A) The apical surface of the endodermal cells bears numerous microvilli. The supranuclear cytoplasm contains many larger vesicles or vacuoles with a small amount of electron dense content. (B) Invaginations (arrowheads), small coated vesicles (cv) and tubules are present in the most apical regions of the cytoplasm. Scale bars: 4 μm (A); 1 μm (B). Discussion As in other hystricomorph rodents [ 3 , 4 ], the placenta of paca consists of several lobes separated by interlobular trophoblast. The center of each lobe contains maternal arteries from which blood flows to the periphery through the trophoblastic channels of the labyrinth. In the fetal capillaries, blood flows from the periphery towards the center, allowing for countercurrent exchange [ 5 , 6 ]. The interlobular regions are made up of cords of syncytiotrophoblast, which define maternal blood spaces. We show here that each interlobular region drains several lobes. In the labyrinth, the trophoblast is bathed directly by maternal blood and is separated from the fetal capillaries by a single layer of syncytiotrophoblast. Thus the placental barrier is syncytial haemomonochorial, as in the guinea pig [ 7 - 9 ], chinchilla [ 10 ], cane rat [ 11 ], degu [ 12 ] and rock cavy [ 13 ]. The apical membrane of this trophoblast, which is in contact with maternal blood, is well supplied with microvilli. There are recesses in the basal membrane and caveolae can occasionally be seen at the apical membrane. However, we did not observe transtrophoblastic channels such as those described in the degu [ 12 ]. In the interlobular region, the trophoblast contained mitochondria and rough endoplasmic reticulum. The surface in contact with maternal blood bore numerous microvilli. The interlobular trophoblast is the functional equivalent of the spongy zone of murid rodents [ 14 ]. In the guinea pig it has been identified as the principal site of progesterone synthesis [ 15 ]. In addition, it is thought to synthesize progesterone-binding protein [ 16 ], which is found in the plasma throughout gestation and is unique to hystricomorph rodents. Much of the surface of the placental disk is covered by the endoderm of the parietal yolk sac. This epithelium is largely columnar and rests on Reichert's membrane. Beneath the membrane are trophoblast cells that differ from those in other regions of the placenta in their larger size, rounder form and vacuolated appearance. An appropriate designation for these cells is spongiotrophoblast. It has been shown in the guinea pig that maternal protein can cross the spongiotrophoblast and Reichert's membrane. It can then pass into the uterine lumen through the intercellular spaces between the endoderm cells, as they are not sealed by tight junctions [ 17 ]. In theory the protein could then be taken up by the visceral yolk sac, but whether maternal-fetal exchange occurs by this circuitous route is open to speculation. The subplacenta is a unique feature of hystricomorph rodents [ 3 ]. Characteristic of the subplacenta of paca were the large intercellular spaces between the cytotrophoblasts and the lacunae within the syncytiotrophoblast. As in the guinea pig [ 18 ] and chinchilla [ 10 ], the lacunae in the syncytium were lined by microvilli and contained electron-dense material. It seems likely that the lacunae intercommunicate, but this requires further investigation. Wolfer and Kaufmann [ 19 ] suggested that the subplacenta might be a highly active area from a metabolic point of view. They pointed out that the structure had been carefully described, but that little was known about its function, except that it might have endocrine activity. Recently it was proposed that the subplacenta is an important source of invasive trophoblast in the guinea pig, chinchilla, capybara and degu [ 20 ]. We found multinucleated giant cells in the junctional zone between the subplacenta and decidua. Intriguingly, the cytoplasm of these cells contained electron-dense granules reminiscent of those found in the subplacental syncytium. The cytoplasm of the giant cells had areas of low electron density, a feature also shared by the subplacental syncytium. These cells were PAS-positive and may store glycogen or glycoprotein. The placenta of paca is attached to the uterus by a prominent structure, formed largely of maternal tissue, that we have denoted the placental pedicle. It was first described by Strahl [ 21 ] and named by him the mesoplacenta. A similar structure occurs in the agouti [ 4 ], chinchilla [ 22 ] and nutria [ 5 ]. The equivalent formation in the capybara and guinea pig placenta is the placental stalk [ 23 ]. Trophoblast is found in the walls of the maternal vessels that pass through the pedicle to supply the placenta [ 4 ]. Like other hystricomorph rodents, paca has an inverted yolk sac placenta that persists until term. This visceral yolk sac displays folds and complex villi. The numerous digitiform projections are sometimes branched. They consist of a mesenchymal axis covered by a simple columnar epithelium of endodermal cells. The cells seem to have a high level of endocytotic activity. Similar characteristics are found in the yolk sac endoderm of the guinea pig [ 24 ], chinchilla [ 10 ] and rock cavy [ 13 ]. In the guinea pig it has been shown experimentally that immunoglobulin G is taken up from the uterine lumen to coated pits. The endocytotic vesicles thus formed are transported to the lateral membrane and empty into the intercellular spaces by exocytosis [ 25 ]. From here the immunoglobulins are presumed to reach fetal capillaries. Protein cannot move directly into the intercellular spaces because of the tight junctions near the apex of the cells [ 25 ]. In addition to this mechanism for conferring passive immunity to the fetus, there is nonspecific uptake of protein from the uterine lumen. Many endocytotic vesicles fuse with larger vacuoles that form part of the cell's lysosomal apparatus [ 26 ]. The protein they contain is thought to provide amino acids to the fetus. Given the similarity in ultrastructure, these mechanisms are likely to operate in the yolk sac placenta of paca. In conclusion, the placenta of the paca conforms to patterns previously described for hystricomorph rodents [ 3 , 14 ]. Common features include the lobulation of the placenta and the presence of a subplacenta. At the ultrastructural level they comprise the haemomonochorial nature of the interhaemal barrier and the pinocytotic apparatus of the visceral yolk sac endoderm. The lobulated structure of the placenta allows for a larger exchange area and the development of precocial young [ 14 ]. Recently it was argued that more attention should be given to the hystricomorph rodents as models in human medicine. They bear a closer genetic similarity to humans than do murid rodents, such as the mouse and rat, because the latter have undergone a very high rate of gene mutation [ 27 ]. Since the paca carries a singleton fetus with a birth weight of 640–900 g, it deserves particular consideration as a potential model of fetal growth and development [ 28 ].	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555959.xml
529460	Effect of Ampicillin on the kinetics of colonization of Streptococcus pneumoniae and Lactobacillus fermentum in the respiratory tract of mice	Ampicillin was selected to further study the effect of this antibiotic on the colonization capability of S. pneumoniae and L. fermentum intranasally inoculated in a mice experimental model. The sensitivity of S. pneumoniae and L. fermentum to antibiotics was evaluated by different "in vitro" techniques. The results showed that both microorganisms have a typical pattern of sensitivity to antibiotics in these assays. The "in vivo" experiments showed that the treatment with Ampicillin increased the number of lactobacilli and neumococci in the groups of mice treated only with one of the microorganisms. In those mice treated with Lactobacillus , challenged later with neumococci and treated with Ampicillin, the pathogen in lung decreased on the 4 th day, disappearing completely after on. The histological studies showed that the antibiotic treatment decreased the inflammatory response produced by the pathogen at the lung and trachea levels.	Introduction Respiratory tract infections are commonly caused by Streptococcus pneumoniae . Extensive antibiotic use for these infections as well as misuse for viral respiratory infections has led to increased penicillin resistance amongst the streptococci [ 1 - 6 ]. Even though there is a very broad description of the pattern of sensibility to antibiotics of this pathogen and other potentially pathogenic microorganisms, there are a small number of publications referred to the lactobacilli sensitivity to these types of compounds [ 7 - 10 ]. The antibiotic treatment modifies the stability of the normal or indigenous microbiota, producing the dominance of certain microorganisms able sometimes to produce a secondary infection [ 11 ]. There are a lot of approaches trying to restore the normal microbiota, or to avoid modifications of the different ecosystems to prevent infections, both for human and animal application. One of the main research areas related to the restoration of the indigenous flora is the application of probiotic microorganisms [ 12 - 14 ]. Lactic Acid Bacteria (LAB) has been widely used to restore the ecologic equilibrium of different areas [ 12 - 14 ], mainly for the gastrointestinal tract. In the last decade, there is a lot of other research areas referred to the potential application of probiotics in the respiratory tract mainly as vaccine vectors [ 15 - 17 ]. They could be applied for the protection against pathogenic microorganisms as S. pneumoniae which is a frequent nasopharynx colonizer. In previous papers, the isolation and identification of the microorganisms of the normal microbiota of the respiratory tract of mice was reported by our research group. Also, the evolution from the moment they were born up to two months age was published [ 18 ]. In the isolated microorganisms, the probiotic or beneficial characteristics were studied, selecting some strains of the genus Lactobacillus that shared some properties [ 19 ]. From them, a strain of Lactobacillus fermentum was selected by the beneficial probiotic properties. Later the optimal dose to produce a transitory colonization or permanence [ 20 , 21 ] and the protection exerted against S. pneumoniae [ 22 ] intranasally inoculated was determined. The objective of the present paper was to study the effect of specific antimicrobial agents against the growth of S. pneumoniae and L. fermentum by "in vitro" assays. Also, to study the effect of Ampicillin, orally administered in a mice experimental model, against the intranasal inoculation of S. pneumoniae , L. fermentum or both microorganisms. The "in vivo" assays were complemented with histological and cytological studies to determine if there was some type of general response of the animals to the treatment. Materials and methods Microorganisms and culture media L. fermentum was isolated from the respiratory tract (pharynx) of adult BALB/c mice [ 18 , 20 - 22 ]. The mutants resistant to Rifampicin (RR) were obtained to differentiate the inoculated strains from the normal microbiota. The conditions of storage and culture were described previously [ 18 , 20 - 22 ]. S. pneumoniae A6 serotype was isolated from human pneumonia-suffering subjects, and identified by standard techniques. The serotypification was performed at the "Servicio de Bacteriología Clínica, Instituto Nacional de Enfermedades Infecciosas-ANLIS "Dr Carlos G. Malbran, Buenos Aires, Argentina by Quellung technique. Pathogenicity in mice was increased by inoculating S. pneumoniae intraperitoneally [ 22 ]. The pathogen was stored in 25% glycerol added to BHI broth (Brain-Heart Infusion) at -70°C. Antibiotics assayed those antibiotics broadly used for the treatment of Gram Positive microorganisms affecting the respiratory tract were studied by determining the Minimal Inhibitory Concentration: penicillin, ampicillin, ceftriazone, ceftazidine, claritromicine, tetracicline, rifampicin, ciprofloxacin, aztreonam, cloramphenicol and imipenen. Quantitative determination of the bactericidal activity of antibiotics against lactobacilli The Minimal Inhibitory Concentration Method (MIC) recommended by the National Committee for Clinical Laboratory Standards (NCCLS) was used, replacing the culture media by MRS agar as basal media [ 23 ], because Lactic acid bacteria are not able to grow in the recommended Muller Hinton agar. The reference strains used were Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 . Antibiotic sensitivity of S. pneumoniae . Method of diffusion and dilution in agar The behavior of the pathogenic microorganisms with antimicrobial substances was determined by the qualitative and quantitative techniques of diffusion and dilution in agar (MIC), according to the National Committee for Clinical Laboratory Standards, 2001 (NCCLS). The antibiotics assayed for the qualitative method were oxacillin, vancomycin, chloramphenicol, tetracycline, rifampicin, ciprofloxacin, trimetroprim-sulphametoxazol, claritromycin. The antibiotics assayed for the quantitative method were penicillin, ampicillin, ceftriazone and claritromycin. The reference strain used was S. pneumoniae ATCC 49619. "In vivo" assays Each experimental group (or assay) included 24 to 30 animals. Four to six mice were killed in each experimental day. Each one of the "in vivo" experiments was performed twice, and their data used to calculate the media and Standard Deviation. The protocols used were accepted by the Animal Ethics committee of CERELA. L. fermentum and Ampicillin assay groups of adult (two months old) male BALB/c mice were intranasally inoculated with 4 doses of a RR L. fermentum strain every 12 h. (50 μl of a suspension containing 1 × 10 9 CFU/ml). Later, Ampicillin was administered by the oral way in 4 doses of 100 mg/Kg/day, fractionated in two daily doses (1.5 mg/dose/mice) every 12 h. The experimental protocol is schematized in Fig 1a . Figure 1 Experimental protocols, doses of microorganism and time schedule applied to perform the "in vivo" assays: Fig 1a: Administration of L. fermentum (empty arrows) and Ampicillin (A). Fig 1b : Administration of S. pneumoniae (red arrow) and Ampicillin (A). Fig 1c: Administration of L. fermentum , S. pneumoniae and Ampicillin. The days when mice were sacrificed are indicated with black crosses. S. pneumoniae and Ampicillin assay BALB/c mice were intranasally inoculated with a dose of 50 μl of a suspension of S. pneumoniae in saline solution (1 × 10 9 CFU/ml). The antibiotic was administered by the oral way in 4 doses of 100 mg/Kg/day, following the same scheme than before. The experimental protocol is schematized in Fig 1b . L. fermentum + S. pneumoniae + Ampicilin assay Mice were intranasally inoculated with RR L. fermentum (4 doses of 50 μl of a suspension of 1 × 10 9 CFU/ml), then challenged with S. pneumoniae A6 by the same way (50 μl of a suspension of 1 × 10 9 CFU/ml) and later treated with Ampicillin in four doses every 12 hours as before, as shown in Fig 1c . All the experimental groups were sacrificed by cervical dislocation on days 2, 4, 7 and 10 post antibiotic administrations. The samples from nasopharynx and pharynx were obtained with a cotton swab. Trachea, bronchia and lung were aseptically removed and homogenized with a Teflon pestle. The number of microorganisms was determined by the successive dilutions method with peptone water, and plated in MRS agar-Rifampicin. The plates were incubated for 24 h. at 37°C in microaerophilic environment. The identification of microorganisms was performed by macroscopic, microscopic characteristics, Gram staining and biochemical tests. Histological studies the samples for histological assays were obtained from the higher trachea, located at the neck basis, bronchia in the area where the main bronchia are divided, and lung in the terminal bronchiole area and alveolar wall. They were fixed with 10% paraformaldehyde and stained with Hematoxylin-eosin and Ramon and Cajal technique [ 24 ]. They were analyzed by light microscopy. Cytological technique The left lobule of the lung was used to perform the cytological slices. The organ was first immersed in saline solution, then fixed in methanol for 3 min, and afterwards stained with the Romanovsky method (Giemsa stain-Merck,Germany) used routinely in the lab [ 25 ]. Statistical techniques The numbers in the figures represent the mean and Standard Deviation of the results obtained in the two set of experiments performed for each assay. The Student's t test was applied to determine the differences statistically significant of the data. Results Antimicrobial sensitivity L. fermentum The MIC results for L. fermentum (ug/ml) are as follows: penicillin, ampicilin, ceftriazone, ceftazidine, rifampicin, ciprofloxacin: ≥100 ug/ml, tetraciclin, imipenem and aztreonam: 10 ug/ml, cloramphenicol 1 ug/ml, claritromicine: 0.1 ug/ml. Comparing these values with those obtained for the MIC of Enterococcus spp and Streptococcus spp , type strains recommended by NCCLS, L. fermentum would be resistant to penicillin, ampicillin, ceftriazone, rifampicin and ciprofloxacin. S. pneumoniae The results of the antimicrobial sensibility of S. pneumoniae are shown in Table 1 and 2 , demonstrating by the two methods applied that S. pneumoniae is sensitive to all the antibiotics assayed by both, the disc diffusion assay, and the MIC. Table 1 Sensitivity of S. pneumoniae to different antimicrobials by the disc diffusion assay Antibiotic Concentration (ug) Diameter (mm) Results R (1) S (2) Oxacillin 1 - ≥20 24 Vancomycin 30 - ≥17 22 Chloramphenicol 30 ≤20 ≥21 24 Tetracycline 30 ≤18 ≥23 32 Rifampicin 5 ≤16 ≥19 28 Ciprofloxacin 5 ≤13 ≥17 29 Trimetoprim+Sulphametoxazol 1.25 + 23.75 ≤15 ≥19 26 Claritromicin 15 ≤16 ≥21 25 (1) Resistant, (2) Sensitive. Table 2 Sensitivity of S. pneumoniae to antimicrobials by the Agar dilution method (MIC) Antibiotic MIC (ug/ml) Break point Sensitive Results (ug/ml) Penicillin - ≤0.12 ≤0.02 Ampicillin - ≤0.25 ≤0.10 Ceftriazone ≥2 ≤0.50 ≤0.10 Claritromicin ≥1 ≤0.25 ≤0.02 "In vivo" assays from the results obtained in "in vitro" sensitivity assays, that are predictive, the antibiotic Ampicillin was selected to be used in " in vivo " assays at the recommended dose for human beings (equivalent to the one applied to respiratory tract infections therapy). Mice treated with L. fermentum (1 × 10 7 CFU/ml/dose) and Ampicillin Mice inoculated intranasally with four doses of lactobacilli (determined previously as the dose needed to obtain colonization of the respiratory tract) and treated with Ampicillin showed an increased colonization of lactobacilli during all the days studied in nasal and pharynx exudates (p < 0.01), with values of 10 7–8 CFU/organ on days 1 and 4, and lower values on the following days. The higher number were in nasal and pharynx exudates. In the control mice without antibiotic, Lactobacillus numbers were around 10 6 CFU on the first days of the assay. On the 7 th and 10 th days with antibiotic treatment, Lactobacillus were still present in all the organs, except in lung, while in the mice without antibiotics they were only present on nasal and pharynx exudates on day 7 disappearing on day 10 (upper right Figure 2 ). The results obtained are shown in Figure 2 . Figure 2 Colonization of L. fermentum in the respiratory tract of mice obtained after the administration of Ampicillin. The figures express the number of L. fermentum in nasal instillations, pharynx, trachea, bronchia or lung from mice inoculated intranasally with L. fermentum (4 doses of 10 7 CFU/mouse) and treated with 4 doses of Ampicillin. The inserted figure shows the number of lactobacilli obtained from control mice without antibiotic. () indicates differences statistically significant between the two groups of mice Mice challenged with S. pneumoniae (1 × 10 7 CFU/ml) and treated with Ampicillin Mice challenged with the pathogen and treated with the antibiotic showed that neumococci were present a longer time in all the organs of the respiratory tract, (as shown in Figure 3 ) compared with the respective mice without antibiotic treatment (upper right figure 3 ). The differences statistically significant (p < 0.01) between the two groups of mice are indicated in the figure. Figure 3 Colonization of S. pneumoniae in the respiratory tract of mice obtained after the administration of Ampicillin. The figures express the number of S. pneumoniae in nasal instillations, pharynx, trachea, bronchia or lung from mice, challenged with S. pneumoniae (10 8 CFU/mouse) and treated with 4 doses of Ampicillin. The inserted figure shows the number of pathogens obtained from control mice without antibiotic. () indicates differences statistically significant between the two groups of mice Mice treated with L. fermentum , S. pneumoniae and Ampicillin The colonization of Lactobacillus in this group was similar to the one obtained in the experimental group treated with Lactobacillus and antibiotics without the pathogen (not showed results) In the case of S. pneumoniae , the previous treatment with Lactobacillus , reduced significantly the number of pathogenic microorganism in the lung, as showed in Fig 4 . It was present only on the first day, being completely cleared on the following days studied. Figure 4 Colonization S. pneumoniae in mice inoculated intranasally with L. fermentum (4 doses of 1 × 10 7 CFU/dose), challenged with S. pneumoniae (1 × 10 8 CFU/mice) and treated with 4 doses of Ampicillin compared with mice treated only with the pathogen and antibiotics. The figures express the number of pathogen in lung. Histological and cytological studies The histological studies showed that in all the organs of the respiratory tract there are not significant structural modifications. Only a moderate leukocyte exudation is observed in the alveolar region in the mice treated with S. pneumoniae a nd Ampicillin These results are resumed in Table 3 . The administration of Lactobacillus produces a stimulation of the lung macrophages, and a lymphocytic infiltration at the trachea level, as resumed in Table 3 and Figure 5a . Mice treated with lactobacilli, S. pneumoniae and antibiotic decreased the inflammatory response produced by the pathogen (picture 5b) compared with the pattern produced by the pathogen and antibiotic, represented in Fig 5c . Table 3 Histological modifications of the respiratory tract of mice on day 4 of the experiment. Organ control L.f .+ampicillin (1) S.p .+ampicillin (2) L.f .+ S.p .+ampicillin (3) Bronchia Cilindric epithelia Conserved Conserved Conserved Bronchioli Cubic epithelia Conserved Without extension of the interalveolar wall Conserved Lungs Alveolus and duct with plane epithelia. Regular duct and alveolus. Alveolar light with macrophages Regular exudation of mononuclear lymphocytes close to the interaveolar duct. Slight areas with a scarce increased lymphocytic density Mice were inoculated with: L. fermentum + ampicillin (1), S. pneumoniae + ampicillin (2) and L. fermentum + S. pneumoniae + ampicillin (3). Figure 5 Light microscopy photographs (200×) of histological slices stained with hematoxylin-eosin from mice intranasally treated with L. fermentum , (5a) S. pneumoniae (5b) or both (5c) and later with Ampicillin. Fig 5a: regular ducts (long arrow) and alveolus (short arrow) (100×), Fig 5b: regular mononuclear exudation close to the interalveolar duct (100×). Fig 5c: Increased lymphocytic density (100×). The cytological studies showed an increased number of activated macrophages by Lactobacillus , neumococci , or both. The addition of the antibiotic produces an activation of the non-specific line of defense represented by the alveolar macrophages, as shown in Table 4 . Table 4 Cytology of lung impressions stained with Giemsa on day 4 post inoculation in mice treated with L. fermentum + S. pneumoniae +ampicillin Normal Macrophages G1 Macrophages G2 Macrophages Lymphocytes Neutrophils Other cells Control Mice 26 +/- 2 13 +/- 4 10 +/- 2 25 +/- 4 9 +/- 3 17 +/- 5 L. fermentum + ampicillin 14 +/- 4 30 +/- 5 28 +/- 4 14 +/- 3 9 +/- 2 5 +/- 4 S. pneumoniae +ampicillin 18 +/- 3 25 +/- 5 30 +/- 2 7 +/- 4 12 +/- 2 8 +/- 5 L.f + S.p . + ampicillin 20 +/- 2 30 +/- 2 25 +/- 3 13 +/- 2 6 +/- 2 6 +/- 3 Discussion One of the main goals of our research is to go further into the mechanisms involved in the probiotic effect in the respiratory tract [ 26 , 27 ]. Having in mind that we have available a mice experimental model, our interest was focused in the knowledge of the effect produced by the antibiotics more frequently used to treat the respiratory infections on the kinetics of colonization of both microorganisms, either separately or combined. We were also interested in the effect produced by antibiotics on the microbial colonization by the potential use of probiotics together with antibiotics as therapeutic agents and for the restoration of the normal microbiota. Our results showed that the administration of Ampicillin by the oral route and Lactobacilus intranasally increases their colonization in the respiratory tract. These results support the use of antibiotics together with probiotics, which would help in having a higher colonization of the protective microorganisms. A paper published by Dielemen et al [ 28 ] demonstrated that the administration of Lactobacillus GG prevents recurrence of colitis in transgenic rats after the treatment with Vancomycin and Imipenem, showing that our hypothesis has been proved in the intestinal tract. Coincidently with these results, the treatment with Ampicillin, even though the S. pneumoniae strain used was sensitive to Ampicillin in the "in vitro" test, in the experiments performed in mice produces an increase in the number of the pathogenic microorganism that are also present a longer time in the tract. The pathogen produces a lymphocytic infiltration al the tracheal level, together with an increased inflammatory response evidenced in the lung cytological studies. Why the response to the pathogen is different in "in vitro" assays than in "in vivo" experiments?. Referred to the behavior of Lactobacillus , there is a broad discussion based on the different results obtained through the application of "in vitro" test, and the differences found in "in vivo" assays. Even though the first one can be used as screening and to predict some characteristics or properties assayed in experimental animals models, one must consider that not always they are coincident [ 29 ]. In those experiments performed in mice treated with Lactobacillus , neumococci and Ampicillin, the final results is that the pathogen was cleared faster from the lung supporting the combined use of lactobacilli and antibiotics in the prevention of the infections produced by this pathogen. These results are also demonstrated by the histological slides, (Fig 5c ) where the effect produced by the pneumococci decreased by the concomitant use of lactobacilli and antibiotics, when compared with the damage produced by the pathogen administration (Fig 5b ). The histological modifications produced by the pathogen in mice previously protected with lactobacilli is lower compared to that obtained when the pathogen alone is inoculated into mice, [ 20 ] which produces congestion zones and edema in the terminal bronchiolar area. The activation of lung or alveolar macrophages produced by the administration of L. fermentum , S. pneumoniae , or both, indicates the highly active non-specific branch of the immune system, which is an important first line of defense against microbial invasion in the lower airways infection. This activation could also help in the clearance of the neumococci observed on the 4th day of the experiments. These results do not agree with those from one infections produced by Pseudomonas aeruginosa , that does not produce an activation of the Pulmonary Alveolar Macrophages [ 30 ]. The activation of the immune system at the respiratory tract is taking more relevance, mainly by some researchers who study this way of administration of different antigens and vaccines [ 15 - 17 ]. More studies must be undertaken trying to elucidate which are the mechanisms involved in the protection exerted by lactobacilli in the respiratory tract, and also which are the reasons of the increased colonization of the pathogen and lactobacilli by the effect of the Ampicillin treatment. Authors' contributions Rosa Cangemi de Gutiérrez, carried out the experimental and microbiological assays in animals Viviana Santos, performed the histological and cytological studies Marta Cecilia, participated in the interpretation of antibiotic assays Clara Silva, carried out the in vitro sensibility test María Elena Nader-Macías: conceived the study and participated in its design, evaluation of the results and writing of the manuscript	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC529460.xml
322746	In Methuselah's Mould	Is aging inevitable or can it be "cured"? Recent work from many different fields of science is now providing clues into why we age and how long we might live	The pathologist makes do with red wine until an effective drug is available, the biochemist discards the bread from her sandwiches, and the mathematician indulges in designer chocolate with a clear conscience. The demographer sticks to vitamin supplements, and while the evolutionary biologist calculates the compensations of celibacy, the population biologist transplants gonads, but so far only those of his laboratory mice. Their common cause is to control and extend the healthy lifespan of humans. They want to cure ageing and the diseases that come with it. “I would take resveratrol if it were feasible,” notes David Sinclair, assistant professor of pathology at Harvard Medical School in Boston, Massachusetts. In the meantime, he adds, “I do enjoy a glass of red wine about once a day.” It was Sinclair's laboratory, in association with a commercial partner, that revealed last August how the team had identified for the first time a group of simple organic molecules capable of extending lifespan. The most proficient of the group is resveratrol, the plant polyphenol found in red wine, and its discovery as a potential elixir to combat ageing represents another extraordinary advance in a decade of discoveries that have revolutionised the field. “These molecules will be useful for treating diseases associated with ageing, like diabetes and Alzheimer's.” Extending Life Although the life-enhancing effects of Sinclair's polyphenols are so far confined to the baker's yeast Saccharomyces cerevisiae , the work suggests that researchers are only one small step from making a giant leap for humankind. “People imagined that it might have been possible, but few people thought that it was going to be possible so quickly to find such things,” says Sinclair. The field of ageing research is buzzing. Resveratrol stimulated a known activator of increased longevity in yeast, the enzyme Sir-2, and thereby extended the organism's lifespan by 70% ( Box 1 ). Sir-2 belongs to a family of proteins with members in higher organisms, including SIR-2.1, an enzyme that regulates lifespan in worms, and SIRT-1, the human enzyme that promotes cell survival ( Figure 1 ). Though researchers still do not know whether SIRT-1, or “Sir-2 in humans,” as Sinclair puts it, has anything to do with longevity, there is a good chance that it does, judging by its pedigree. In any event, resveratrol proved to be a potent activator of the human enzyme. This might not be altogether surprising, at least not now, given that the polyphenol is already associated with health benefits in humans, notably the mitigation of such age-related defects as neurodegeneration, carcinogenesis, and atherosclerosis. Figure 1 SIRT-1 Deacetylase—the Human Enzyme That Promotes Cell Survival—in a Dividing Human Cell The enzyme is marked in red, and the image is superimposed on acetylated proteins (green) and condensed chromosomes (blue). (Image courtesy of David Sinclair.) “The study came out from a pretty big gamble,” recalls Sinclair, who used the human enzyme to screen and identify molecules that he expected would also stimulate those related enzymes in lower organisms. Unlike SIRT-1, these related enzymes are known to increase longevity when activated, usually by restricting the organism's calorie intake. Not only did they find “a whole collection of related polyphenols that activate ‘Sir-2 from humans,’ … but we put them onto yeast, justbeing the simplest model, and amazingly [they] did what we were hoping [they] would do,” recalls Sinclair. “But it was a real long shot.” Now there's great eagerness in the Sinclair laboratory to complete and publish related research, notably by replicating the yeast work in higher organisms. “We have very promising results in Drosophila , which is a huge jump from a yeast cell,” says Sinclair. “So we're very encouraged by that.” Publication of these results is imminent. The team has also quickly broadened its horizons and is already testing the polyphenols on mouse disease models. “We think we may have tapped into a cell survival and defence programme [and] that these molecules will be useful for treating diseases associated with ageing, like diabetes and Alzheimer's,” says Sinclair. He hopes to publish the diabetes results by mid-2004 and those for Alzheimer's by the end of the year. Harvard and BIOMOL Research Laboratories, its commercial partner based in Pennsylvania, have already filed a patent application for the use of “synthetic related molecules” to combat diseases of ageing—an application, Sinclair adds, “very much linked to the [polyphenols] paper.” There's been a radical shift in attitude towards ageing, says Sinclair. Before the 1990s, “people thought that we were a lot like cars, that we would just rust and breakdown—nothing we could do about it. The new idea is that there are pathways that can boost our defences against ageing—the ‘ageing-can-be-regulated’ discovery … that genes can control ageing [and] that there are pathways that [we can use to] slow down the process,” he says. “If that's true—and it really seems to be true for a lot of organisms—if it's true for us, it really means that there is hope that we will be able, one day, to find small molecules that can alter these pathways.” How Long Could We Live? Sinclair expects to see such developments within his lifetime, but he ridicules the notion that humans will experience anything like the 70% extension to lifespan of his cultured yeast. “It'll be great if we can just give people an extra five years and have less disease in their old age and make it less painful,” he says. “We won't be seeing any Methuselahs around,” he insists. On his side are James Vaupel, one of Europe's leading demographers, and Marc Mangel, a mathematical modeller at the University of California at Santa Cruz. “Since 1840, life expectancy has been going up at 2.5 years per decade and will continue at this rate, maybe a little faster,” says Vaupel, head of the Laboratory of Survival and Longevity at the Max Planck Institute for Demographic Research in Rostock, Germany. Women in Japan currently have the highest average life expectancy of 85, he notes: “So the figure could be 100 in six decades, but not 500.” There's remarkably little people can do even if they want to live as long as possible, he says. “Give up smoking, lose weight, don't drive when drunk, install a smoke detector, take regular exercise,” suggests Vaupel, who insists he does them all, as well as taking vitamin supplements. “You look at these worms and think, ‘Oh my God, these worms should be dead.’ But they're not. They're moving around.” Mangel sees the problem of assessing the limitations of ageing research as fairly straightforward. Mathematical models, he says, could solve it by linking demographic properties and physiological developments. “We've had a separation of the biology of ageing and the demography of ageing, and they need to come together again,” notes Mangel, whose personal anti-ageing regime involves taking “a dose of anti-oxidant chocolate with a good feeling.” But Cythnia Kenyon, whose laboratory reported in October that it had generated a 6-fold increase in the lifespan of its nematodes, is not so sure about the limitations. “You look at these worms and think, ‘Oh my God, these worms should be dead.’ But they're not. They're moving around…. Once you get your brain wrapped around that … then you start thinking, oh my goodness, so lifespan is something you can change—it's plastic. Then who knows what the limit is?” (Cynthia Kenyon has recorded video clips of the superstars of her lab, Caenorhabditis elegans , to show how long-lived mutant nematodes are as vigorous as normal young adults [ Videos 1–4 ].) Warming to the theme, Kenyon hypothesises: “If you'd asked me many generations ago, when we were actually common precursors of worms and flies, ‘Cynthia, you have a two-week lifespan, do you think that you could [live longer]?’ And if I'd told you, ‘Well, I think our descendants will live 1,000 times longer,’ you'd have said, ‘Oh, come on!’ But we do. It happened,” she notes. “Who knows what you could do in people?” Kenyon muses. “I don't want to go on record saying that it's not possible in people because I don't see why it wouldn't be…. I'm certainly not imagining that my company in the next few years is going to come up with a compound that can make people live to be 500. That seems just preposterous.” So the timescale is millions of years? “No, not necessarily,” she insists, “because once we understand the mechanism, then we can intervene and see what we can accomplish.” Box 1. Model Systems for Ageing Yeast, as a model system for ageing, is at a distinct disadvantage. It lacks an endocrine system, and yet much research indicates that the key to longevity is control of hormones such as insulin and insulin-like growth factor 1 (IGF-1), as well as their downstream pathways and associated tissues, including the reproductive network. But David Sinclair, whose laboratory used yeast to show how an elixir might extend life, remains sanguine. “If it doesn't have an endocrine system, we can't understand cell-to-cell communication, but not all of ageing is just communication,” he says. “There are things that occur inside the cells that provide longevity, and that's where yeast can be applied.” Sinclair, assistant professor of pathology at Harvard Medical School, discovered a group of polyphenols that cause the human enzyme SIRT-1 and its homologues in lower organisms, including Sir-2 in yeast, to deacetylate the p53 protein and its homologues, notably the histones H3 and H4, in yeast. “Our findings are that the activation of the pathway downregulates p53's ability to cause cell death,” he notes. Although p53, the tumour suppressor, is known to be involved in programmed cell death, it is not known whether SIRT-1 has any role in ageing. So Sinclair “went straight back to yeast to prove the principle of longevity extension.” He found that deacetylisation of histones in yeast caused the DNA that's wrapped around them to become more compact and thus more stable. “DNA stability is key to longevity, and Sir-2 promotes that,” says Sinclair. “We don't know yet whether it's the same in humans.” Cynthia Kenyon, meanwhile, sees worms as the optimal model for helping to substantiate the links to humans. Besides the nematodes' being multicellular organisms with endocrine systems, she also notes that their short lifespan of around 20 days is a big advantage: “You can do lots of experiments with them.” Mice, which have short lifespans for mammals, still live two years, and long-lived mice for three or four years, she notes. And the advantage of fruit flies? “It's good to use more than one animal.” Kenyon, professor of biochemistry and biophysics at the University of California at San Francisco, has focused on decoding the role of genes in ageing, notably daf-2 , whose receptor is similar to those for insulin and IGF-1 in humans and inhibits ageing, and daf-16 , which promotes it. “The DAF-2 receptor activates a highly conserved PI-3-kinase, the PDK/Akt pathway, and that pathway affects ageing, at least in part, by inhibiting the activity of the DAF-16 transcription factor,” says Kenyon. “It does so by phosphorylating DAF-16 and inhibiting its entry into the nucleus.” She adds: “We think that the DAF-2 pathway has another way of influencing ageing … but we don't know what this other way is.” In the long-lived mutants, which are defective in the daf-2 receptor gene or in the genes encoding downstream signalling components, such as the PI-3-kinase, DAF-16's activity in the nucleus leads to the changes in expression of a wide variety of downstream genes, between 100 and 200, estimates Kenyon. Her studies show, she says, that a large number of those genes influence ageing. Signalling Life and Sweet 16 Kenyon, professor of biochemistry and biophysics at the University of California at San Francisco, is among the key contributors responsible for showing that a single gene, and subsequently many genes, can change an organism's lifespan. “It is inconceivable … that a life-extending therapy will ever be developed that is able to extend life independent of every other change.” In a seminal paper published a decade ago, Kenyon's laboratory showed that mutations in the daf-2 gene doubled the lifespan of the nematode C. elegans . daf-2 encodes a receptor that is similar to those for insulin and insulin-like growth factor-1 (IGF-1) in humans; this hormone receptor normally speeds up ageing in worms, but the mutations inhibit its action and enable the organisms to live longer. Before the results appeared, there was a “very negative attitude” towards ageing research, recalls Kenyon. Since then, and especially over the past few years in response to later findings, graduate students have been scrambling for a chance to work in her laboratory. “You can't believe the difference—there was such resistance to it,” she says. “ daf-2 made a huge difference.” But then so did her subsequent research in the field. Among her most significant findings is the identification of many more longevity genes; the results, published in July, derive directly from her early work on daf-2 . “We discovered that in order for long-lived worms to live so long, they need another gene called daf-16 ,” recalls Kenyon. “ daf-16 is kind of the opposite of daf-2 , in the sense that it promotes longevity and youthfulness … so we call it ‘sweet 16.’” daf-16 encodes a transcription factor that controls the expression of more than 100 genes. “They don't do just one thing, they do many things,” says Kenyon. They can act as anti-oxidants (to prevent damage from oxygen radicals), as chaperones (to prevent misfolded proteins from forming aggregates), as antimicrobials (to protect against bacteria and fungi), and as metabolic agents. “So the picture that emerges is that the way the insulin/IGF-1 hormone system produces these enormous effects on lifespan is by coordinating the expression of many genes that do different things to affect lifespan, each of which on its own has only a small effect,” notes Kenyon. “It's as though daf-2 and daf-16 , the regulators, would be the conductors of an orchestra. They bring together the flutes and the violins and the French horns, each of which do different things, and they make them all work together in concert.” Kenyon is unequivocal about the bottom line: “Now we have a whole set of genes whose biochemical functions we can be working on to understand more about the actual mechanisms of ageing.” Complementary results in flies and mammals persuade her to be more explicit. “The common ancestor of worms, flies, and mice must have had an insulin/IGF-1-like hormone system that controlled ageing. And that ability has been maintained. So the question is, has [that ability] been lost in humans? I think it's quite likely that it will also function in humans, but there isn't a direct demonstration yet that that's the case.” Nevertheless, the discoveries about the role of the insulin/IGF-1 pathway in ageing have had a profound impact on her own lifestyle, which includes a tendency to discard the bread from sandwiches and eat only the toppings of pizzas ( Box 2 ). “I'm on a low-carb diet. I gave my worms glucose, and it shortened their lifespan. [The diet] makes sense because it keeps your insulin levels down,” she says. “Caloric restriction extends lifespan of mice, and so does the insulin/IGF-1 pathway,” she notes. Indeed, starting a low-calorie diet at any point in adulthood appears to help fruit flies live longer, according to research in Britain published last September. “What we don't know for sure in mice,” Kenyon continues, “is whether the two pathways are different or the same.” While much ageing research focuses on these two influences, she says that there are another two areas of investigation. Her laboratory reported in December 2002 that inhibiting the respiration of mitochondria in developing worms increased longevity, but that it had no effect in adult worms, for reasons still unexplained, she says. Further microarray analysis is underway to pinpoint whether the cause simply lies downstream of the insulin/IGF-1 pathway or whether it is something different altogether. The Price of Life Then there's research looking at the effects on lifespan of changes to an organism's reproductive system. For Kenyon, such work often involves a battle to convince sceptics that longevity is not a trade-off with fertility. Four years ago, her laboratory reported that killing germ cells increases the lifespan of worms by 60%, but only because, she stresses, it affects endocrine signalling and not because it prevents reproduction. Further research, published last year, showed quite clearly, she says, that ageing and reproduction are controlled independently of one another. And as for her recent work on infertile worms, which lived six times as long as normal following the removal of their entire reproductive systems, she says: “If we could intervene in the hormone signalling pathways directly, we think the animals would still live six times as long as normal, but would be fertile as well.” Jim Carey is one of those “trade-off” sceptics. He is a population biologist at the University of California at Davis and his research, on the effect on life expectancy of replacing the ovaries of old mice with ovaries from younger mice, is intended to complement Kenyon's work. But he insists that “an honest discussion of lifespan extension must include consideration of tradeoffs.” Many manipulations that extend lifespan in model systems, whether genetic or dietary, for example, ignore or gloss over the side effects, such as permanent sterility, huge weight loss, distorted organ-to-body ratios, or major behavioural aberrations, he notes. “It is inconceivable to me that a life-extending therapy will ever be developed that is able to extend life independent of every other change,” he concludes. “All life systems are interlinked and hierarchically integrated at all levels, so to talk about life extension using analogies with a car warranty concept is wrong-headed.” Another “trade-off” sceptic takes a different tack. As Armand Leroi puts it: “During occasional periods of involuntary celibacy I have thought, well, I may not be getting laid, but at least I shall live to a miserable and solitary old age.” Leroi, an evolutionary biologist at Imperial College of Science, Technology, and Medicine in London, offers an optimistic appraisal of the chances of finding a cure for ageing in his new book about the effects of genetic variety on the human body. He sees ageing simply as a collection of curable diseases: “There is no obvious impediment to that advance, nothing to make us think that human beings have a fixed lifespan.” Box 2. Practise What You Preach Cynthia Kenyon's eating habits are defined by her ageing research on worms. “There's a lot of these diets … and what they all have in common is low carb —actually, low glycaemic index carbs,” she says. “That's not eating the kind of carbohydrates where the sugar gets into your bloodstream very quickly [and stimulates production of insulin].” No desserts. No sweets. No potatoes. No rice. No bread. No pasta. “When I say ‘no,’ I mean ‘no, or not much,’” she notes. “Instead, eat green vegetables. Eat the fruits that aren't the sweet fruits, like melon.” Bananas? “Bananas are a little sweet.” Meat? “Meat, yes, of course. Avocados. All vegetables. Nuts. Fish. Chicken. That's what I eat. Cheese. Eggs. And one glass of red wine a day.” Kenyon, professor of biochemistry and biophysics at the University of California at San Francisco, has been on her diet for two-and-a-half years. “I did it because we fed our worms glucose and it shortened their lifespan.” But the diet is unproven, she cautions, and she's not recommending it for all. Nevertheless, she's pleased with its performance for her. “I have a fabulous blood profile. My triglyceride level is only 30, and anything below 200 is good.” Kenyon is angered by the general lack of nutritional knowledge: “It's a little bit embarrassing to say that scientists actually don't know what you should eat…. We can target particular oncogenes, but we don't know what you should eat. Crazy,” she says. Does her dieting represent a return to scientists experimenting on themselves? “I don't think so—you have to eat something, and you just have to make your best judgement. And that's my best judgement. Plus, I feel better. Plus, I'm thin—I weigh what I weighed when I was in college. I feel great —you feel like you're a kid again. It's amazing.” Video 1 Normal Nematodes at Day 1 of Adulthood (Video used by permission from Cynthia Kenyon.) Video 2 Long-Lived daf-2 Mutants at Day 1 of Adulthood (Video used by permission from Cynthia Kenyon.) Video 3 Normal Nematodes at Day 13 of Adulthood The worm on the left is dead. (Video used by permission from Cynthia Kenyon.) Video 4 A Long-Lived daf-2 Mutant at Day 13 of Adulthood (Video used by permission from Cynthia Kenyon.)	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC322746.xml
529448	Gastro-enteritis outbreak among Nordic patients with psoriasis in a health centre in Gran Canaria, Spain: a cohort study	Background Between November 2 and 10, 2002 several patients with psoriasis and personnel staying in the health centre in Gran Canaria, Spain fell ill with diarrhoea, vomiting or both. Patient original came from Norway, Sweden and Finland. The patient group was scheduled to stay until 8 November. A new group of patients were due to arrive from 7 November. Methods A retrospective cohort study was conducted to assess the extent of the outbreak, to identify the source and mode of transmission and to prevent similar problems in the following group. Results Altogether 41% (48/116) of persons staying at the centre fell ill. Norovirus infection was suspected based on clinical presentations and the fact that no bacteria were identified. Kaplan criteria were met. Five persons in this outbreak were hospitalised and the mean duration of diarrhoea was 3 days. The consequences of the illness were more severe compared to many other norovirus outbreaks, possibly because many of the cases suffered from chronic diseases and were treated with drugs reported to affect the immunity (methotrexate or steroids). During the two first days of the outbreak, the attack rate was higher in residents who had consumed dried fruit (adjusted RR = 3.1; 95% CI: 1.4–7.1) and strawberry jam (adjusted RR = 1.9; 95% CI: 0.9–4.1) than those who did not. In the following days, no association was found. The investigation suggests two modes of transmission: a common source for those who fell ill during the two first days of the outbreak and thereafter mainly person to person transmission. This is supported by a lower risk associated with the two food items at the end of the outbreak. Conclusions We believe that the food items were contaminated by foodhandlers who reported sick before the outbreak started. Control measures were successfully implemented; food buffets were banned, strict hygiene measures were implemented and sick personnel stayed at home >48 hours after last symptoms.	Background On 5 November 2002, the Department of Infectious Disease Epidemiology at the Norwegian Institute of Public Health (NIPH) was contacted by a dermatologist at Rikshospitalet, a tertiary care university hospital in Oslo, Norway. He reported several cases of gastro-enteritis in a medical care centre in Argúinegúin, Gran Canaria, Spain where Nordic patients with psoriasis undergo climate therapy. The symptoms (diarrhoea and vomiting) were reported to be of short duration (1–2 days). The patient group was scheduled to stay until 8 November. A new group of patients were due to arrive from 7 November. An outbreak management team was created in order to investigate and control the current outbreak, and to prevent similar problems in the following group. The outbreak management team consisted of investigators working in public health institutes in the countries involved (Sweden, Finland, Norway and Spain). The investigation was organised and co-ordinated from the NIPH in Oslo, Norway. The objectives of this investigation were to assess the extent of the outbreak, identify the mode of transmission, the vehicle and the causative pathogen and recommend appropriate control measures. Methods The health centre is a private institution owned by the Norwegian Asthma and Allergy Association. Every three weeks the health centre receives a group of 100–110 Nordic patients suffering from skin diseases (mainly psoriasis). Rikshospitalet, Oslo, Norway administers the project. Patients are residents of Norway, Sweden and Finland. On site, Nordic medical staff (one dermatologist, three nurses and a sport and leisure leader) provides health services for the patients. The centre also hosts private individuals and employees of the Asthma and Allergy Association. We conducted a retrospective cohort study among all Nordic patients attending dermatological care and employees staying at the health centre between 2 and 10 November. The persons from the Asthma and Allergy Association, and employees eating and staying outside the centre in the same time period were not included since there were no reported cases among this group and they did not eat or use the same facilities as the dermatological patients. Case definition A case was defined as a person who 1) attended dermatological care or worked at the Health Centre in Gran Canaria, 2) took meals at the Centre and 3) fell ill between the 2 and 10 November 2002, with symptoms of diarrhoea (3 or more loose stools in 24 hours), vomiting or both. Recruitment of the cohort Health personnel at the health centre provided a guest list with name, address, age, sex and bungalow number. For cases they also recorded date of onset of symptoms and stool sampling and results of microbiological analysis. A standard questionnaire was developed at the Norwegian Institute of Public Health, translated in each national centre and mailed to the entire cohort, with instructions to fill it in and send it back to the public health institute in their country of origin. The local health authorities in Gran Canaria enquired about gastro-intestinal illness in the community. They contacted health centres, churches and schools in the area. Exposure An exposure was defined as a food item eaten or an environmental factor present within the 2 days before onset of illness (the number of cases and the number of persons in the denominator therefore varies for different days of analysis). Possible exposures included consumption of all food items served in the health centre's restaurant, consumption of food served outside the centre, brushing teeth in tap water, drinking tap water, consumption of ice cubes, swimming in one of the two pools, swimming in the ocean, having had contact with an ill person, hand washing habits before meals, and the sharing of bungalow with symptomatic persons. We recorded the number of times food items were eaten to enable the study of potential dose-response relationships. Analysis Day by day, we compared food-specific attack rates (AR) for each item for the exposed and the non-exposed. Suspecting the etiological agent to be norovirus and knowing it has a short incubation period (24–48 hours) [ 1 ] only food consumption in the two days preceding onset of symptoms were considered as possible vehicle of the infection. Therefore, when considering food eaten on 31 October, only cases that became ill 2 November were in the numerator. Cases ill on or after 3 November were included in the denominator. When considering food eaten on 1 November, cases that became ill on 2 and 3 November were in the numerator. Likewise, persons were removed from the data set after they were reported ill, since they were no longer at risk. Then we pooled the results for the two first days of the outbreak, assuming that person-to person transmission would be low these first days. We used EpiData software (Epidata Association, Denmark) for data collection and analysed them with SPSS version 10.0 (SPSS Inc. Chicago, Illinois). Attack rates, relative risk and 95% confidence intervals were calculated for each of the food items and other exposures. Only variables with RR greater than 2.0 are presented in this report. A multivariable analysis was run to assess potential confounding, including age, gender and the pooled variables with RR greater than 2.0 in the univariable analysis. Laboratory and environmental investigations All cases in the cohort presenting gastro-enteric symptoms were encouraged to deliver a stool sample. The samples were sent to a local private laboratory in Gran Canaria. Both virological and bacterial tests were requested. Food sampling of some of the served items was performed on 5, 21 and 22 November by the local health authorities in Gran Canaria. The health authorities also took samples on 12 November from water taps and from the pools. Both stool and environmental samples were to be sent to Madrid for virology testing. Due to misunderstandings the samples were never forwarded to Madrid. Virological analyses were therefore not performed. Results We mailed 110 questionnaires to the patients who stayed at the Centre between 17 October and 8 November 2002, and employees (n = 6) eating at the centre. Ninety-one questionnaires (response rate = 78%) were returned (2 from employees, 89 from patients at the centre). Personal characteristics Among the 91 respondents there were 47 men and 44 women. The median age was 48 years (range 18–80). Forty-eight persons fulfilled the case definition (attack rate (AR) = 53%). The AR was not significantly different by genders or nationalities. The AR was higher among those above 70 years of age (80%). Temporal distribution The outbreak peaked on November 4, with 16 cases (Figure 1 ). The outbreak extended from 2 to 7 November. In addition, two kitchen workers, without dates of onset, were ill with diarrhoea just before the outbreak started. None of the other staff eating outside the centre were reported ill. No cases were reported among private individuals and employees of the Asthma and Allergy Association staying in the same compound, but who ate only outside the health centre. Disease characteristics Forty-two cases had diarrhoea and 33 vomited (Table 1 ). The mean duration of symptoms was 3,7 days for diarrhoea (range 1–23 days) and 1 day for vomiting. Twenty-four cases reported having seen a doctor in Gran Canaria. Seven consulted a doctor after returning home. Five persons went to hospital and 11 received intravenous treatment. Six had to stay home from work on average for 1 day. Ten of the respondents reported 24 possible secondary cases among persons they had been in contact with after they returned home. Cases were distributed among most of the bungalows. The AR for those sharing room with two others were 42% (39/93), AR for those sharing room with one person 38% (3/8) and AR among those 9 living in a single room was 44%. Two cases were employees living private or in a separate section. Six cases fell ill subsequent (more than 10 hours later and within two days) to their roommate. There were no gastro-enteritis cases reported in the community concurrently to the outbreak in the health centre. Cohort study, food specific attack rates From the daily analysis of food consumption, strawberry jam, dried fruit (eaten on both 1 and 2 November) and butter (eaten on 31 October) were associated with risk of illness (RR> 2.0) for those who became ill on 2 and 3 November (Tables 3 and 4 ). For those ill 4 and 5 November eating pear was associated with risk of illness (RR 2.8, 95% CI: 0.6–14.4) and eating strawberry jam (RR 1.2, 95% CI: 0.4–3.5). None of these items were independent associated with disease onset 4 and 5 November, in the multivariable analysis. When pooling food consumption 1 and 2 November, eating pear and drinking full milk also increased the risk of developing gastro-enteritis. In the multivariable analysis including sex, age group, and all variables with RR>2.0, consumption of dried fruit (adjusted RR = 3.1, 95% CI: 1.4–7.1) and strawberry jam (adjusted RR = 1.9, 95% CI: 0.9–4.1) were independently associated with disease (Table 3 ). AR for those with who became ill on November 2 and 3 increased with the amount of strawberry jam eaten. No dose response was observed for dried fruits (Table 4 ). Laboratory results and aetiology We reviewed the clinical symptoms: 69% vomited, 85% had < 72 h duration of illness and all stool samples were found negative for bacteria. Based on this, norovirus was suspected as the aetiological agent. This hypothesis is supported by Kaplan criteria (Table 2 ) [ 2 ]. Stool samples from 6, non symptomatic food handlers were taken on 8 and 12 November and repeated on 28 November. Two of the food handlers were reported to be symptomatic just prior to the outbreak. All stool samples taken from employees and patients staying at the centre (altogether 43 stool samples) were reported negative for bacteria at the private laboratory in Gran Canaria. Virological analyses were not performed. Six food items and water from the two pools were analysed for bacteria: chicken croquettes, frozen chicken breast and leg, sausage, cheese and salmon. Results were negative. Discussion We initiated an epidemiological investigation of an outbreak of gastro-enteritis in a health centre in Gran Canaria, Spain. Based on Kaplan criteria, our findings suggest that the aetiological agent was norovirus. Results of the cohort study suggests that the outbreak was initiated by ingestion of dried fruits, strawberry jam or both, followed by person-to-person transmission. There is no supporting microbiological or environmental evidence. Pathogenesis and mode of transmission Norovirus outbreaks can often be diagnosed presumptively on clinical grounds from their characteristic epidemiological features [ 2 ]. Kaplan has reported four criteria that indicate with a high sensitivity and a relatively high specificity that a gastroenteritis outbreak is caused by norovirus [ 3 ]. In this outbreak all four criteria were met. The Kaplan criteria were used since a confirmatory microbiological diagnosis was pending. We collected time of onset of symptoms, but not of recovery. We therfore had to use 0–72 h cut off instead of 12–60 as in the Kaplan criteria. We do not belive this affected our results. A high proportion of persons in this outbreak reported that they received IV-treatment (n = 11, saw a general practitioner (n = 24), were hospitalised (n = 5) and the mean duration of diarrhoea was 3.7 days. The reported consequences, especially the duration of the illness was severe compared to many other norovirus outbreaks [ 3 - 5 ]. The explanation may be that many of the cases suffered from chronic diseases and were treated with drugs reported to affect the immunity (methotrexate or steroids). This explanation is supported by two recent reports where norovirus gastroenteritis is described not to be so mild in certain groups in the community [ 6 ] and in hospital patients' [ 7 ]. We believe there were two modes of transmission: a common source for those who fell ill on 2 and 3 November and thereafter mainly person to person transmission. Low RR for all food items at the end of the outbreak and the short incubation period for noroviruses, together with the fact that norovirus outbreaks usually have high rates of person-to-person transmission [ 8 ], support this hypothesis. We did however not find a higher attack rate among those who shared room or bungalow. Only nine persons had a single room. There were no cases among those persons who stayed at the health centre, but did not eat in the health centre restaurant or in the community during the outbreak. This suggests a foodborne outbreak with its origin at the centre's restaurant. Vehicles of contamination Dried fruits, strawberry jam or both were the probable vehicles of contamination. Strawberry jam and dried fruits were handled and kept at the kitchen in the Health Centre and not supplied by the catering service. They were served in buffet style on plates. Both food items are biologically plausible vehicles. Contaminated hands or silverware could explain contamination of these food items, but these modes of transmission were not verified. One hypothesis was that the food items were contaminated by the foodhandlers who reported being ill before the outbreak started. Both foodhandlers were involved in preparing the food for the buffet. Very few organisms of norovirus are needed to transmit the disease [ 4 ]. The jam was commercial and cooked. To our knowledge, none of these specific food items has been incriminated as a vehicle in norovirus outbreaks reported in the literature. There are, however, several similar food products that have been involved in norovirus outbreaks [ 1 , 8 , 9 ]. Dose-response analyses give further support for contamination of the strawberry jam. Those who ate jam twice doubled their risk of developing gastro-enteritis. Method Suspecting norovirus, with a short incubation period, as the causal agent [ 1 ], we treated each day as a new cohort. We assumed that persons falling ill at the beginning of the outbreak were infected by a common source, while those persons falling ill later could have been infected by person-to-person transmission, by a common source, or both. When looking at the whole period as a cohort and thus looking at all persons falling ill between 2 and 10 November, then, none of the exposures seemed to increase the risk of disease. The association between food items and disease was probably masked by a high number of cases infected by person-to-person transmission. Eight cases had not eaten dried fruit during the 2 days before falling ill. The concept of dried fruit is different in Spain from Scandinavia. In Spain, dried fruit means raisins, which were served in the restaurant. In the Nordic countries, dried fruit is in general understood to be a mixture of different types of dried fruits. This difference in concepts may have introduced an information bias. We asked for food history for 5 days. The food was always served buffet-style. The menus included more than 300 different food items. To reduce potential recall bias, we pooled the food items consumed on either of the 2 days prior to onset. The fact that strawberry jam and dried fruit remained associated for those with onset 2 and 3 November (Table 3 ) is an argument against the problems related to recall bias. Intervention Based on clinical suspicions of norovirus infection, NIPH suggested to the centre medical personnel to apply a guideline on control of norovirus infection in hospital care setting [ 10 ]. We recommended implementing these guidelines with a special focus on improved hygiene measures and individually served food instead of buffet meals. The health personnel at the health centre supervised the implementation of the guideline. We recommended taking new stool samples of the kitchen workers that had been ill with gastro-enteritis symptoms and excluding all symptomatic food handlers from work for 48 hour after their first normal stool. We also recommended looking for structural and operational deficiencies in the health centre kitchen and in the catering company. Further environmental investigation by the local public health authorities was recommended. The importance of taking stool samples, and analysing them for both virus and bacterial pathogens, was emphasised. The control measures were successfully implemented. Guidelines to control norovirus for a hospital care setting are more demanding in hygiene measures than guidelines for hotel outbreaks. Taking into account that qualified health personnel were in place, potential cases were more susceptible to the disease because of underlying diseases, so these strict measures were justified. It was not possible to cancel the planned arrival of the persons due at the centre from 7 November. Stopping the group of patients with scheduled arrival in the end of November was discussed. Patients expecting to travel to Gran Canaria got written information about the situation at the centre. As no new cases were reported after 14 November (see epilogue), we recommended that this group should travel as planned. Epilogue Among persons arriving 7 November (n = 100), 18 became ill with gastro-enteritis. There were no cases after 14 November. Four of seven stool samples were positive for Salmonella. This finding did not change the belief that norovirus caused the gastro-enteritis among persons in the outbreak described in this article. Salmonella was not found in any of the 22 stool-samples from our cohort. This, together with the reported symptoms, suggests that there may have been two different outbreaks at the centre during this period. Conclusion Between 2 and 7 November, 66 persons fell ill with diarrhoea, vomiting or both in a health centre for Nordic patients with skin diseases at Gran Canaria. Our data suggest that norovirus caused the outbreak. Our findings suggest that individuals who consumed dried fruit (adjusted RR = 3.1, 95% CI: 1.4–7.1) or strawberry jam (adjusted RR = 1.9, 95% CI: 0.9–4.1) were more likely to contract the disease. One hypothesis is that the food items were contaminated by foodhandlers that had had gastro-enteritis shortly before the outbreak started. Improved hygiene measures and individually served food were successfully implemented. Abbreviations Norwegian Institute of Public Health; NIPH Attack rate; AR Relative risk RR Competing interests The author(s) declare that they have no competing interests. Authors' contributions HME: In charge of the investigation, data handeling and writing of the article PJG: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article. KN: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article. MH: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article. BdJ: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article. AMCR: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article. MK: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article. UD: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article. AGR: Working locally with the outbreak, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article. CM: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article. PA: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC529448.xml
544868	HIV-1 nef suppression by virally encoded microRNA	Background MicroRNAs (miRNAs) are 21~25-nucleotides (nt) long and interact with mRNAs to trigger either translational repression or RNA cleavage through RNA interference (RNAi), depending on the degree of complementarity with the target mRNAs. Our recent study has shown that HIV-1 nef dsRNA from AIDS patients who are long-term non-progressors (LTNPs) inhibited the transcription of HIV-1. Results Here, we show the possibility that nef -derived miRNAs are produced in HIV-1 persistently infected cells. Furthermore, nef short hairpin RNA (shRNA) that corresponded to a predicted nef miRNA (~25 nt, miR-N367) can block HIV-1 Nef expression in vitro and the suppression by shRNA/miR-N367 would be related with low viremia in an LTNP (15-2-2). In the 15-2-2 model mice, the weight loss, which may be rendered by nef was also inhibited by shRNA/miR-N367 corresponding to suppression of nef expression in vivo . Conclusions These data suggest that nef /U3 miRNAs produced in HIV-1-infected cells may suppress both Nef function and HIV-1 virulence through the RNAi pathway.	Background The human immunodeficiency virus (HIV), which infect humans cause acquired immunodeficiency syndrome (AIDS), which has reached pandemic levels in some societies, especially those in Southern Africa and Southeast Asia [ 1 ]. Given the immensity of HIV pandemic, the development of a rather safe and cheap, effective therapeutics, has become the main focus [ 2 ]. Several strategies attempted to control the spread of AIDS have not shown major breakthrough and the vaccines have shown little promise as far as their efficacy is concerned. However, one approach used extensively in other diploid organisms, which now has tremendous potential to encourage antiviral defense against HIV appears to be double stranded RNA-dependent post-transcriptional gene silencing or RNA interference (RNAi). RNAi is a defense mechanism against aberrant transcripts that may be produced during viral infection and mobilization of transposons [ 3 , 4 ]. The RNAi pathway has been implicated in silencing transposons in the C. elegans germline [ 5 , 6 ], silencing stellate repeats in the Drosophila germline, and the response against invading viruses in plants [ 7 ]. Post-transcriptional regulation by RNAi is mediated by small non-coding RNAs (~25-nucleotides; nt). Small interfering RNAs (siRNAs) are short RNA duplexes that direct the degradation of homologous transcripts [ 8 ]. In contrast, the single stranded microRNAs (miRNAs) bind to 3' untranslated regions of mRNA with complementarity of 50 to 85% to give translational repression without target degradation [ 9 ]. The mature miRNA (~25-nt) is produced by processing of ~70-nt precursor stem-loop hairpin RNAs (Pre-miRNA) by Dicer [ 10 , 11 ]. At the moment several human diseases, including spinal muscular atrophy (Paushkin et al ., 2002), fragile X mental retardation [ 13 , 14 ] and chronic lymphocytic leukemia [ 15 ] have been identified as illnesses in which miRNAs or their machinery might be implicated. However, up until now there has been no clear-cut scientific proof that establishes the exact correlation between miRNAs and human infectious diseases such as AIDS. One of the human immunodeficiency virus type 1 (HIV-1) coding accessory genes, nef , is located at the 3' end of the viral genome and partially overlaps the 3'-long terminal repeat (LTR). The nef gene is uniquely conserved in HIV-1, HIV-2 and simian immunodeficiency virus (SIV) and is not essential but important for viral replication in vivo [ 16 ]. The nef gene is expressed during HIV infection and often accounts for up to 80% of HIV-1 specific RNA transcripts during the early stages of viral replication [ 2 ]. Our own investigations have shown that defective variants of nef dsRNA containing the 3'-LTR regions, obtained from long-term non-progressor (LTNP) AIDS patients, actually inhibited the transcription of HIV-1 [ 17 ]. Furthermore, cis -expression of mutated F12-HIV-1 nef inhibits replication of highly productive NL43-HIV-1 strain, which is not related to down-regulation of CD4 [ 18 , 19 ]. It has been demonstrated that F12 nef gene cloned from the provirus of naturally occurring HUT-78 T cells infected with the supernatant of the peripheral blood mononuclear cells (PBMCs) of an HIV-seropositive non-producer patient, induces a block of viral replication [ 19 ]. Thus, it has been suggested that nef RNAs may be a cis -regulatory factor for HIV-1 replication [ 20 ]. In the current study, we have established the link between miRNAs and HIV infections by demonstrating that nef -derived miRNAs are produced in HIV-1-infected cells. The results presented here show that nef short hairpin RNAs (shRNAs) corresponding to the nef miRNAs efficiently block RNA stability or mRNA translation, perhaps an indication that HIV-1 regulates its own replication by using nef miRNAs. Results and Discussion Identification of a candidate of miRNAs in HIV-1-infected cells Very recently, the Epstein-Barr virus (EBV)-encoded miRNAs were identified. Thus, during the preliminary stages of this study, our curiosity was fixed on the need to find out if indeed there was any relationship between nef miRNAs and HIV-1-infected cells. To achieve this purpose, we extracted total RNA from HIV-1 IIIB strain persistently infected MT-4 T cells and northern blot analysis was performed using eight probes against the nef coding region, as shown in Figure 1A . Analyses using several anti-sense probes, small RNA molecules approximately ~25-nt in size were detected as well as HIV-1 major transcripts, 9.1, 4.3 and 1.8 kb bands (Fig. 1B ). Similar results were obtained with total RNA from HIV-1 SF2 strain infected MT-4 T cells (data not shown). RNA samples treated with a mixture of the single stranded specific RNases A and T1 also generated ~25-nt RNAs that hybridized in northern blots with the sense probes against the same nef region. However HIV-1 major transcripts were not detected (data not shown), indicating that the structure of the small RNA molecules could be double-stranded RNAs (miRNAs). Some variability was observed when the quantity of the miRNAs was compared with the total of the major transcripts. A maximum of 3.2% of miRNAs was detected by using #367 probe when compared with total HIV-1 transcripts, and the minimum of 0.3% was detected by using probe# 90 (Fig. 1B ). Figure 1 Detection of HIV-1 nef miRNAs and inhibition of Nef expression by nef RNAs. ( A ) Schematic representation of HIV-1 nef in its genome and synthetic DNA probes (#) used in this study. ( B ) Total RNAs were extracted from MT-4 T cells persistently infected with HIV-1 IIIB, separated on a 15% polyacrylamide-7 M urea gel, and subjected to northern blot analysis. The approximate sizes of the three classes of HIV-1 transcripts and small RNAs are indicated on the right. The loading control was rRNA stained with ethidium bromide. Relative expression (%) of nef small RNAs to the three classes of HIV-1 transcripts is at the bottom of figure. ( C ) Schematic representation of effector plasmids ( E ) H1 promoter-driven shRNA expression plasmids. Reporter (R) Nef-EGFP expression plasmid (pYM2.2) is also shown. (D) Inhibition by sinefs in pH1 plamids of Nef-EGFP expression. Either sinef, siluc or siegfp in each plasmid was transfected into Jurkat T cells in the presence of either pYM2.2 or control pEGFP-N1. At 36 h after transfection EGFP-positive cells were counted by flow cytometry. Data represent the relative activity of EGFP-positive cells where the percentage of positive cells in the sample transfected with pYM2.2 or pEGFP-N1 plus pcDNA3.1 or si(-) in pH1 plasmid was scored as 100%. Data are averages of three independent experiments + SD. Bars, SD. ( E ) Immunoblot analysis showing inhibition of YM2.2 expression by different nef shRNAs. Jurakat T cells were transfected with pYM2.2 and pH1/sinefs or siluc plasmid, cellular lysates were prepared 48 h after transfection, and immunoblotted with rabbit serum against Nef (upper panel) and anti-β-actin antibody ( lower panel ). The β-actin expression shows equal loading of all samples. To randomly clone the nef miRNA, ~25-nt RNAs were gel purified, cloned and sequenced. The sequences from the nef miRNA clones were 5'-acugaccuuuggauggugcuucaa-3' or similar ones, corresponded to the nucleotides approximately 420 to 443 conserved region of nef (miR-N367). The most notable feature of this analysis is that it has proven beyond reasonable doubts that nef -derived miRNAs are produced in HIV-1 infected cells. Inhibition of Nef by plasmids-encoding siRNA/miRNA To examine inhibition of nef expression by the nef miRNA, we constructed eight shRNAs homologous to the native miRNA or probes used in Figure 1A [ 21 ]. Although it has been reported that three to four mutations in the sense strand derived from miRNA could have the potential to control unmutated 21-nt stem loop [ 22 ], we investigated whether the native shRNA-expressing plasmid can effectively reduce nef gene expression or not (Fig. 1C and 1D ). We used egfp or luc gene (pH1/siegfp or luc) as a positive or negative control. All the shRNA-expressing plasmids including the controls were co-transfected into Jurkat T cells with pYM2.2 and cell fluorescence resulting from the expression of EGFP reporter gene was quantified by flow cytometry. The sinef176, 190, 367/miR-N367 and control siegfp all showed efficient reduction, but the sinef 007, 084, 299, 468 and 580 constructs gave only modest reductions, and no suppression was observed with si(-) and luc (Fig. 1D and Table 1 ). Immunoblot analysis using anti-Nef rabbit serum also confirmed the inhibition of Nef-EGFP expression by sinef 176, 190 and 367/miR-N367 (Fig. 1E ). Table 1 Relation between AIDS clinical courses and nef dsRNA or siRNA/miRNA in suppression of nef gene expression Nef inhibitor Nef expressed by: Target region Clinical courses Nef-EGFP HIV-1 IIIB PFV/nef dsRNA * Human SF2 ++ † ++ ND ‡ Full-length (including miR-N367) ND 1-3-3 ++ - ND U3 deleted Rapid progressor, Died within 3 years 4-2-1 ND + ND U3 region (including miR-N367) Rapid progressor, Died within 3 years 15-2-2 ++ +++ ND U3 region (including miR-N367) Non progressor with low plasma viremia 16-1-1 ND + ND U3 deleted Non progressor with low plasma viremia jw95-1 ND + ND U3 region (including miR-N367) Non progressor with undetectable viremia siRNA 15-2-2 model mouse dsnef + + + Full-length ND sinef007 + ND ND Upstream of U3 region ND sinef084 + ND ND Upstream of U3 region ND sinef176 +++ +++ ++ Upstream of U3 region § ND sinef190 +++ +++ +++ Upstream of U3 region ND sinef299 + ND ND U3 region (aa 83–135) ND sinef367/miR-N367 ++ +++ ++ U3 region No weight loss sinef468 + ND ND U3 region ND sinef580 + ND ND U3 region ND dsRNA to LTNPs' nef has been described (Yamamoto et al ., 2002). † -, negative; +, 0 to 50 % inhibition; ++, 50 to 75% inhibition; +++, 75 to 100 % inhibition. ‡ ND, not done. Inhibition of Nef expression by STYLE vector-encoding nef siRNA/miR-N367 To assess the effects of nef miR-N367 in vivo , we constructed a prototype foamy virus (PFV)-based live vector. The PFV vector expressed HIV-1 SF2 nef gene as a reporter and the STYLE vector expressed shRNA as effectors. The full-length nef gene was inserted into the bel-2 portion of a PFV clone (22) in frame to obtain pPFV/nef (Fig. 2A ). The pPFV/nef was transfected into BHK cells and treated with the histone deacetylase inhibitor, trichostatin A (TA) [ 23 ]. The viral supernatant, which contained approximately 5 × 10 6 infectious units (IFU), was collected at 72 h after transfection. For preparation of the STYLE vectors to deliver the shRNAs, the env gene portion of pSKY3.0 was replaced with the shRNA expression cassette under the control of the H1 promoter. The pSTYLE was produced (Fig. 2A ), and transfected into the FFV envelope-expressing packaging cells, CRFK sugi clone # 6, in the presence of TA. The transfected CRFKsugi clone #6 had 99% FFV Env positive cells when analyzed by flow cytometry. The viral supernatant with a titer of ca. 1 × 10 5 IFU was collected at 72 h post-transfection. Figure 2 Inhibition of nef expression in human T cells by nef siRNAs. ( A ) Schematic representation of shRNA-expressing STYLE vector ( E ) and HIV-1 SF2 nef gene expressing pPFV/nef vector (R). The helper plasmid, pFFenv expresses FFV envelope protein under the control of CMV promoter. ( B ) Detection of expression of nef mRNA and integration of vectors. STYLE, SKY3.0, PFV/nef, PFV or mock was used to infect Jurkat T cells and the infected cells were cultured for 2 weeks. After 2 weeks, gag and nef mRNA expression was measured by RT-PCR. Genomic DNA of LTR, gag and nef of STYLE or PFV/nef were also detected by PCR. β- actin was used as a control. ( C ) Inhibition of Nef-EGFP expression by nef siRNA-expressing STYLE in Jurkat T cells. The pYM2.2 was transfected into each of the STYLE or mock-infected Jurkat T cells and EGFP-expressing cells were counted by flow cytometry at 48 h after transfection. Data represent the relative activity of EGFP-positive cells, where the percentage of positive cells in the sample transfected with pYM2.2 upon the STYLE-si(-) infected cells was scored as 100%. ( D ) Inhibition of HIV-1 transcription and replication by nef STYLE-367. HIV-1 IIIB persistently infected MT-4 T cells were transfected with the pLTR SF2 reporter and β-gal expressing control pCMVβ plasmids at 72 h after infection with STYLE. At 48 h post-transfection, Luc activity was measured and normalized as Luc values (Luc/β-gal). Absolute levels of Luc activity in the samples of pLTR SF2 plus SRYLE-si(-) were 16,311 + 1,253 or 783 + 87 light units for STYLE-367/miR-N367 transfectants. Data represent the relative Luc activities where the percentage of positive cells in the samples infected with the STYLE-si(-) was scored as 100%. After 48 h, p24 antigen was also measured in the cell culture supernatant of STYLE-infected Jurkat T cells. Data are averages of three independent experiments + SD. Bars, SD. ( E ) Inhibition of nef expression by nef siRNA in Jurkat T cells. Cells were infected with PFV/nef 48 h after infection with the STYLE and then subjected to semi-quantitative RT-PCR analysis. Data represent the relative expression of mRNA, where the percentage of positive cells in the sample of mock-infected cells (E: Mock) relative to the PFV/nef (R: PFV/nef) infected cells was scored as 100%. Data averages were derived from three independent experiments + SD. Bars, SD. The expression of viral mRNAs and integrated DNAs from either the PFV/nef or STYLE vectors was confirmed by infection of Jurkat T cells. The mRNAs and genomic DNA were extracted from the infected cells at 2 weeks post-infection. The PFV/nef-expressed gag and nef mRNAs and the STYLE-expressed gag mRNA were detected after amplification of these regions using reverse transcription (RT)-PCR. The integration of the DNAs into the genome of Jurkat T cells was also confirmed by PCR of the LTR, gag and/or nef regions (Fig. 2B ). The control SKY3.0 and PFV-infected cells were both negative for nef mRNA and integrated DNA (Fig. 2B ). The integrated DNA was also detected by southern blot analysis with genomic DNA of either PFV/nef or STYLE-infected cells (data not shown). Expression of shRNAs (~22-nt) was also confirmed in STYLE-infected cells by northern blot analysis (data not shown). Expression of Nef protein in PFV/nef-infected cells was also detected with specific rabbit anti-Nef serum in immunoblots (data not shown). To evaluate whether the STYLE encoding siRNA could inhibit the expression of the nef gene in cultured human T cells, pYM2.2 was transfected into each of the STYLE-infected Jurkat T cells (m.o.i. = ca. 0.1). The most efficient sinef176, 190 and 367/miR-N367 vectors for reduction in nef expression (Fig. 1D and 1E ) were selected for this experiment. The EGFP-positive cells were counted by flow cytometry at 48 h after transfection. Expression of Nef-EGFP fusion protein was reduced drastically following treatment with either the STYLE-176 (74 + 3.2) or 190 (51 + 4.2) and also reduced with 367/miR-N367 (32 + 2.3%). Reduction was insignificant with either the STYLE-si(-) (0 + 0.7) or STYLE-luc (7 + 0.9%) controls (Fig. 2C ). The in vitro inhibitory effects of STYLE encoding nef siRNA on HIV-1-infected cells were evaluated in Luc assays and using MT-4 T cells persistently infected with HIV-1 IIIB. Cultivation of the STYLE infected cells for 72 h followed by transfection with the pLTR SF2 and culture for another 48 h showed that STYLE-176, 190 and 367/miR-N367 all significantly (p < 0.005) suppressed Luc activity when compared to controls (Fig. 2D ). HIV-1 p24 Gag was also significantly inhibited in the culture supernatant by infection with STYLE-176, 190 and 367/miR-N367 when compared to controls (p < 0.001) (Fig. 2D ). These data suggested that shRNA/miR-N367 could inhibit HIV-1 transcription and replication in intact HIV-1-infected human T cells. Jurkat T cells that had been transduced with nef shRNA for 48 h were infected with the PFV/nef. Semi-quantitative RT-PCR analysis revealed that while treatment with STYLE-190 dramatically reduced the expression of both nef and gag mRNAs of the PFV/nef, the expression of nef mRNA was also drastically suppressed by STYLE-176 and 367/miR-N367 (Fig. 2E ). However the STYLE-si(-) and luc controls showed ~10% suppression of nef and ~20% suppression of gag mRNAs (Fig. 2E ), which was probably a result of interference following super-infection. Nonetheless, both nef transcription and PFV/nef replication were substantially inhibited by STYLE-176, 190 and 367/miR-N367. Inhibition of Nef expression by siRNA/miR-N367 in mice Since different host gene products are required for siRNA-mediated RNAi and miRNA-mediated translational repression with let-7 and lin-4 in C. elegans , the two RNAs may not have the same functions in vivo [ 24 ]. To test this point, we investigated the efficacy of miR-N367 using STYLE-367 in mammalian tissues. The study mice were group 1 = PFV/nef-infected (n = 6); group 2 = PFV/nef and control STYLE-luc infected (n = 6); group 3 = PFV/nef and STYLE-367-infected (n = 8); and group 4 = STYLE-367-infected (n = 6). Identical study groups were used for both Balb/c and C3H/Hej mouse strains. Nef protein expressing lymphocytes were quantified by histochemical analysis using F3 Nef monoclonal antibody (mAb) or anti-Nef rabbit serum 2 days after PFV/nef infection. Nef protein was detected by immunofluoresence assay in the subcapsular area of the spleens of groups 1 or 2 Balb/c mice, but not groups 3 or 4 (Fig. 3A and Table 2 ). No positive cell staining was observed using normal rabbit serum as a primary antibody (Fig. 3A ). To test the expression of nef , nested RT-PCR was also done on day 2 to evaluate the degree of nef mRNA expression in the spleen, liver, adipose tissues and hematopoietic cells in groups 1–4. The nef mRNA was significantly expressed in liver and hematopoietic cells of Balb/c mice in groups 1 and 2, but not in the group 3 animals that were STYLE-367 infected (Table 2 ). Tissues from group 4 did not show any nef bands after RT-PCR (data not shown). Figure 3 In vivo effects of miR-N367. ( A ) Distribution of Nef positive staining cells in the subcapsular area of groups 2 or 3 mouse spleens at 2 days after infection with PFV/nef. Anti-Nef rabbit serum or normal rabbit serum was used as a primary antibody. ( B ) Immunofluorescence for 305 mAb positive staining cells in the subcapsular area of groups 2 or 3 mouse spleens at 2 days after infection with PFV/nef and immunoperoxidase staining by 305 mAb in cells of interfollicular area of HIV-1 uninfected human spleen and tonsillar follicle. ( C ) Short term body weights of PFV/nef-infected Balb/c mice. The body weights of the PFV/nef-infected mice (group 1, n = 6, solid circle), the PFV/nef-infected followed by the STYLE-luc-infected mice (group 2, n = 6, solid triangle), the PFV/nef-infected followed by the STYLE-367-infected mice (group 3, n = 8, open triangle) and the STYLE-367-infected mice (group 4, n = 6, open circles) were measured from days 0 to 5. ( D ) Long term body weights of PFV/nef-infected C3H/Hej mice. Treatment of each group and numbers of mice were same as ( C ). Bars, SD. ; p < 0.05, *; p < 0.01 (relative to group 3). ( E ) Immunoperoxidase staining by 305 mAb and anti-Nef rabbit serum in cells of mouse or human adipose tissue. Arrows show positively stained areas. Magnification, X 20 (A and B); X 20 and X 200 ( E ). Table 2 Histochemical detection and RT-PCR amplification of Nef from human and mouse tissues Tissues Histochemistry RT-PCR † F3 Anti-Nef rabbit serum 305 nef gag PPARγ Human (HIV-1 uninfected) Spleen - § - + - - ‡ ND Tonsillar follicle - - + - - ND Liver - - + - - ND Adipose tissue (Salivary gland) - - + - - + Bone marrow - - + - - ND Bronchi - - + - - ND Thyroid gland - - + - - ND Heart muscle - - + - - ND Prostate gland - - + - - ND Testis - - + - - ND Colon mucosa - - + - - ND Lung - - + - - ND Adrenal gland - - + - - ND Brain (Cerebrum cortex) - - + - - ND Mouse Group 1 and 2 Spleen + + + + + ND Liver ND ND + + + ND Hematopoietic cells ND ND + + + ND Adipose tissue (Intestine) + + + - - - Mouse Group 3 Spleen - - + ± ± ND Liver ND ND + - - ND Hematopoietic cells ND ND + - - ND Adipose tissue (Intestine) - - + - - + *Histological analysis was performed with human or each group of mouse tissues by using F3 anti-Nef mAb, anti-Nef rabbit serum or 305 mAb as a primary antibody. For secondary antibody, FITC or peroxidase-conjugated antibody was used. † HIV-1 nef , PFV gag ( ‡ HIV-1 gag for human tissues), and PPARγ mRNA expression were detected by RT-PCR with mRNA from human or each group of mouse tissues. § +, positive; -, negative; ND, not done. Because extracellular Nef is internalized into human and mouse lymphocytes and macrophages [ 25 - 27 ], we examined putative Nef receptor molecule (Ner) expression with 305 mAb [ 27 ] in both mouse and human tissues by histochemical analysis. In mice, 305 mAb positive lymphocytes were detected in the subcapsular area of the spleens by immunofluoresence assay (Fig. 3B ) and liver and hematopoietic cells (Table 2 ) in groups 1, 2 and 3, indicating that detection of antigen by 305 mAb was not altered by Nef expression. In HIV-1 uninfected humans, the 305 mAb positive cells were detected by immunoperoxidase staining in spleen (red pulp), tonsillar follicle (germinal center), liver (Kupffer cells), salivary gland (germinal center and adipose cells), bronchi (smooth muscle cells), lung (stroma cells), thyroid gland (colloid), heart muscle (smooth muscle cells), prostate gland (smooth muscle cells), colon mucosa (intestinal absorptive and muscle cells), testis (basement membrane of tubuli seminiferi), adrenal gland (adipose cells), and brain (cerebrum cortex and cortical cells) (Fig. 3B and Table 2 ). Since Nef suppressed PPARγ expression and reduced fatty acid levels in vitro [ 29 - 32 ], we monitored the expression of PPARγ mRNA and body weights of mice. Significant PPARγ mRNA expression in intestinal adipose tissue of group 3, but not group 1 and 2, was detected on day 2 (Table 2 ). All Balb/c mice in group 1 showed sedation and a drastic loss of weight from days 1 to 3 (day 1, p = 0.003; day 2, p = 0.021; day 3, p = 0.032 relative to mice in group 3) (Fig. 3C ). Similar results were obtained in group 2 (Fig. 3C ). However, group 3 mice infected with STYLE-367 did not appear to be sedated and had no drastic loss of weight (Fig. 3C ). The group 4 animals, which were not infected with the PFV/nef but treated with STYLE-367, had no changes in either behavior or weight (Fig. 3C ). In longitudinal examinations done during the post-infection period, the animals in groups 1 and 2 had recovered the lost weight (Fig. 3D ). Similar results were obtained in group 2 from day 1 to 5 (day 1, p = 0.037; day 3, p = 0.044; day 5, p = 0.048 relative to mice in group 3) in the C3H/Hej mouse groups (Fig. 3D ). To assess the above in vivo results, expression of nef mRNA was examined in adipose tissues (Table 2 ). As shown in Table 2 , although mRNAs of nef and gag were not detected in mouse adipose tissues, 305 mAb and anti-Nef rabbit serum positive staining cells were detected in mouse group 1 and 2 adipose tissues (Fig. 3E and Table 2 ). Considering that the 305 mAb positive staining adipocytes appeared in mouse as well as human tissues (Fig. 3E and Table 2 ), these data suggest that the interaction between 305 and soluble Nef detected in adipose tissues may be responsible for the weight loss observed in mice. In this study, whereas siRNA has been reported to inhibit hepatitis B virus replication in vivo (33–34), our results show that nef -derived miRNAs are produced in HIV-1 infected cells, and support the possibility that miRNA and siRNA may be functionally identical, at least in a retrotransposon such as HIV. Recent studies have revealed that miRNAs and siRNAs could block mRNA expression by similar mechanisms [ 9 ] and that siRNAs could function as miRNAs [ 35 ] and EBV-encoded miRNAs were found [ 36 ]. Our results reported here are consistent with these previous observations and are suggestive of the fact that nef miR-N367 could regulate nef expression even in vivo . In our unpublished data, HIV-1 LTR promoter activity was inhibited by miR-N367 (nt number 379 to 449 of SF2 nef , 71-nt) expression, of which activity was dependent on negative responsive element (NRE) of U3 region (our unpublished data). Although no mismatch shRNA against region #367 was active, the miR-N367 from HIV-1 genome may have some mismatches and effectively inhibit HIV-1 transcription. Further the effects of siRNAs of Tc 1 , in particular those to the terminal inverted repeats derived from read-through transcription of entire transposable elements, were presented for silencing transposase gene expression by RNAi machinery in germ lines of C. elegans , [ 37 ]. Taken together, it could be implied from these and our other results that miRNAs produced in HIV-1-infected cells can efficiently block not only Nef function but also HIV-1 replication through RNAi, which renders persistently low pathogenic infection latent as observed in an LTNP of 15-2-2 (see Table 1 ). It is equally important that although the weight loss reported here occurred only temporarily in vivo , however the inserted nef gene in the foamy retrotransposon may represent miRNAs which could inhibit nef mRNA expression by presumably an identical mechanism to that observed of siRNAs. Thus, RNAi might serve as a new sequence-specific therapeutic arsenal in AIDS prevention and possibly treatment. Overall, our results indicate that nef shRNA transduced into T cell line inhibited HIV-1 transcription. Further, nef miRNAs could be produced from infected T cells and can block the trans-activity of Nef as well as HIV-1 replication on its own via the cis-action of nef. These functions of nef via RNAi pathways may allow persistently low pathogenic or latent infection as observed in HIV-infected non-progressors. Cumulatively, these data suggest that Nef may be involved in both viral replication and the disease progression, the findings, which may facilitate new strategies for HIV control in vivo . Materials and Methods Patient details Patient selection is showed in Table 1 . These SF2 (HIV-1 subtype B prototype) was included as a control nef sequence, because of the inclusion of viruses, which were also subtype B. The SF2 contained full-length nef reading frame as indicated in Table 1 . Patients 1-3-3, 4-2-1 (Table 1 ) are rapid progressors infected with HIV-1. These patients were infected in 1984–1985 and died within 3 years of primary infection with >1 × 106 viral copies and CD4+ T cell count of 75 and 110/ml blood. Patients 15-2-2 and 16-1-1 (Table 1 ) are slow progressors, who were infected in 1984 and have survived HIV-1 infection with high and stable CD4+ T cell counts (690 and 760/ml blood) with low (<5000 copies) plasma viremia. All these patients acquired virus through homosexual sex. JW95-1 (Table 1 ) is a boy who was infected from his mother via breast feeding. The child was infected in 1983 and has survived disease free with high CD4+ T cell count (890/ml blood) with undetectable viremia. Human samples were obtained from a donor after informed consent. Cells and viruses HeLa and BHK cells were grown in Dulbecco's modified Eagle Medium (DMEM) (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. CRFK cells were grown in Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO) with 10% FBS and antibiotics. Jurkat T cells and MT-4 T cells persistently infected with HIV-1 IIIB strain were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% FBS and antibiotics. The packaging cells (CRFKsugi) were made by transfecting CRFK cells with 10 μg of the pFFenv with Lipofectin Reagent (Invitrogen) and selecting transformants after culture for 14 days with 25 μg/ml of hygromycine B (Invitrogen). After 14 days, FFV Env protein expression was measured by flow cytometry and immunoblot analyses with FFV-infected cat B serum [ 34 ]. The pPFV/nef (10 μg) was transfected into BHK cells and pSTYLE/si (10 μg) was transfected into CRFKsugi cells with Lipofectin Reagent. The transfected cells were cultured for 72 hr, and the viral supernatant was collected and filtered through a 0.45 μm pore size Millex-GP filter (Millipore, Bedford, MA). Vector stocks were stored at -70°C prior to use. Viral titers were determined as described previously [ 21 ]. Cells were infected with PFV/nef and/or SKY/si at an m.o.i. of ca. 0.1 in the presence of 4 μg/ml of polybrene and infected cells were cultured at a density of 1 × 10 6 cells per ml for 3 days. The details of plasmid constructs and the primer sequences used in cloning strategies are shown in supplementary file (see Additional file: 1 ). Flow cytometric analysis Flow cytometry was performed with a FACS Calibur (Becton Dickson, San Jose, CA) as described previously [ 17 ]. Luc assay and Immunoblotting Firefly Luc assay was performed using the Luciferase Assay System (Promega) as described previously [ 17 ]. Immunoblotting was performed essentially as described previously by Otake et al. [ 28 ]. P24 ELISA The concentration of p24 supernatant was determined by an antigen capture assay (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions. Confocal laser microscopy analysis Confocal laser microscopy analysis was performed as described previously [ 28 ]. Northern blot analysis Total RNAs were extracted from HIV-1 IIIB or SF2 persistently infected or uninfected MT-4 T cells using TRIzol reagent (Invitrogen). Approximately 40 μg of total RNA was treated with RNase A and T1 (Sigma, St. Louis, MO) as described previously [ 17 ], subjected to electrophoresis on a 15% polyacrylamide-7 M urea gel and electroblotted to HybondN+ (Pharmacia, Uppsala, Sweden) for 4 hr at 400 mA. RNAs were immobilized by UV crosslinking and baking for 1 hr at 80°C. Hybridization was done with an ECL direct Kit (Pharmacia). Synthetic DNA probes were labeled with horseradish peroxidase. The sequence for synthetic sense DNA probes for northern blot analysis are as follows: #007 (5'-gcgtcgacggcaagtggtcaaaacgta-3'); #084 (5'-gcgtcgacgccagcagcagatggggtg-3'); #176 (5'-gcgtcgacgtgcctggctagaagcaca-3'); #190 (5'-gcgtcgacgcacaagaggaggaga-3'); #299 (5'-gcgtcgacgactggaagggctaatttg-3'); #367 (5'-gctcgacggctacttccctgattggc-3'); #468 (5'-gcgtcgacggtagaagaggccaatgaa-3'); #580 (5'-gcgtcgacgcatttcatcacatggccc-3'). RNAs were cloned by 5'RACE System (Invitrogen, CA., USA) with a slight modification in that primers were used that were the same as the synthetic DNA probes as described above that are abbreviated as #primer. In brief, gel purified small RNAs were annealed with #primer and first strand cDNA was synthesized with SuperScript II RT (Invitrogen, CA., USA). Afrer RNase H and T1 treatment, a homopolymeric tail was added to the 3'-end of the cDNA using terminal deoxynucleotidyl transferase and dCTP. After ethanol precipitation, PCR amplification was done with abridged anchor primer and #primer. Then the PCR products were obtained using abridged universal amplification primer and #primer. The PCR fragments were digested with Sal I and cloned into Sal I site of pBluescript SK(-), followed by sequence analysis. The secondary structures of RNAs were predicted by GENETYX-MAC program (Software Development Co. Ltd, Tokyo, Japan). Semi-quantitative RT-PCR analysis Semi-quantitative RT-PCR analysis was performed using the ThermoScript RT-PCR System (Invitrogen, CA., USA) according to the manufacturer's protocol with the following primers: III (5'-atcatgggccaaagagaattc-3') and IV (5'-aaatttcactcaatcgagcc-3') for FFV LTR, VI (5'-aggacctgaaaggcatg-3') and VII (5'-ttgttgagatcgtcccg-3') for FFV gag , VIII (5'-tgtggtggaatgccactag-3') and IX (5'-attgtcatggaattttgta-3') for PFV LTR, XI (5'-tcttacagaccagtaacaa-3') and XII (5'-gtcaatcattacatctgca-3') for PFV gag , XIII (5'-aactactagtacccttcagg-3') and XIV (5'-aaaactcttgctttatggcc-3') for HIV-1 gag , XV (5'-atgggtggcaagtcaaaacg-3') and XVI (5'-tcagcagtctttgtagtactccg-3') for HIV-1 nef , XVII (5'-gttatgggtgaaactctgggagat-3') and XVIII (5'-atgttcctgaacataatcgtc-3') for PPARγ , XIX (5'-gacaacggctccggcatgtgcaaag-3') and XX (5'-ttcacggttggccttagggttcag-3') for β- actin , respectively. The nested PCR followed RT reaction was performed as described previously [ 34 ]. PCR products were quantified with the NIH image program. Relative mRNA expression was calculated as percentage expression using the following formula: integrating number of nef or gag bands/integrating number of β- actin X 100. In vivo studies and tissue analyses Balb/c and C3H/Hej mice were raised under specific pathogen-free (SPF) conditions. Mice were infected with 1 ml of 10 5 IFU of PFV/nef and STYLE-367 by intravenous (i.v.) injection. RT-PCR analyses were performed 2 days after infection. For histological analysis, cryostat sections were prepared from both human and mouse tissues. The fixed sections were rinsed with PBS and incubated with 5% BSA for at least 1 hr to inhibit nonspecific binding of antibodies. Sections were incubated overnight at 4°C with anti-Nef rabbit serum, F3 or 305 mAb, and incubated with peroxidase or FITC conjugated secondary antibodies. The washed sections were incubated in 0.03% 3,3'diaminobenzidine (Sigma) solution in 0.05 M Tris buffer with 0.01% H 2 O 2 for development of peroxidase activity. After counterstaining with hematoxylin or methylgreen, the sections were dehydrated and mounted. Statistical methods Data were analysed using a one-way ANOVA analysis with a post-hoc Fisher's test. P values of 0.05 or more were determined for that of cut off. List of abbreviations used HIV-1 human immunodeficiency virus type 1 miRNA microRNA nt, nucleotides LTNP long-term non-progressors shRNA short hairpin RNA AIDS acquired immunodeficiency syndrome RNAi RNA interference siRNA small interfering RNA LTR long terminal repeat SIV simian immunodeficiency virus PBMCs peripheral blood mononuclear cells; EBV Epstein-Barr virus; EGFP enhanced green fluorescence protein PFV prototype foamy virus TA trichostatin A IFU infectious units FFV feline foamy virus RT reverse transcription m.o.i., multiplicity of infectionm Ab monoclonal antibody; Ner Nef receptor molecule; NRE negative responsive element. Competing interests The authors declare that they have no competing interests. Authors' contributions S.O. carried out northern analyses, immunoblot analyses, RNAi assays and was involved in the construction of plasmids. M.I. and Y.T. participated in in vivo studies and tissue analyses. Y.I. and H.O. participated in data validation and overall experimental design. E.A.B. and N.K.S. carried out the clinical, sequencing, and virological studies and the writing of the manuscript. Y.R.F. participated in the design of the study and coordinated it. All authors read and approved the final manuscript. Supplementary Material Additional file 1 Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544868.xml
524178	A comprehensive evaluation of food fortification with folic acid for the primary prevention of neural tube defects	Background Periconceptional use of vitamin supplements containing folic acid reduces the risk of a neural tube defect (NTD). In November 1998, food fortification with folic acid was mandated in Canada, as a public health strategy to increase the folic acid intake of all women of childbearing age. We undertook a comprehensive population based study in Newfoundland to assess the benefits and possible adverse effects of this intervention. Methods This study was carried out in women aged 19–44 years and in seniors from November 1997 to March 1998, and from November 2000 to March 2001. The evaluation was comprised of four components: I) Determination of rates of NTDs; II) Dietary assessment; III) Blood analysis; IV) Assessment of knowledge and use of folic acid supplements. Results The annual rates of NTDs in Newfoundland varied greatly between 1976 and 1997, with a mean rate of 3.40 per 1,000 births. There was no significant change in the average rates between 1991–93 and 1994–97 (relative risk [RR] 1.01, 95% confidence interval [CI] 0.76–1.34). The rates of NTDs fell by 78% (95% CI 65%–86%) after the implementation of folic acid fortification, from an average of 4.36 per 1,000 births during 1991–1997 to 0.96 per 1,000 births during 1998–2001 (RR 0.22, 95% CI 0.14–0.35). The average dietary intake of folic acid due to fortification was 70 μg/day in women aged 19–44 years and 74 μg/day in seniors. There were significant increases in serum and RBC folate levels for women and seniors after mandatory fortification. Among seniors, there were no significant changes in indices typical of vitamin B 12 deficiencies, and no evidence of improved folate status masking haematological manifestations of vitamin B 12 deficiency. The proportion of women aged 19–44 years taking a vitamin supplement containing folic acid increased from 17% to 28%. Conclusions Based on these findings, mandatory food fortification in Canada should continue at the current levels. Public education regarding folic acid supplement use by women of childbearing age should also continue.	Background Neural tube defects (NTDs) are birth defects resulting from the failure of neural tube closure during early development of the human embryo. The 1997 Canadian national NTD birth prevalence was 0.75 per 1,000 births (live births and stillbirths), down from 1.16 per 1,000 in 1989 [ 1 ]. The rates tend to be higher in the eastern provinces than in the west [ 2 - 4 ]. Historically, Newfoundland has had one of the highest rates in North America with a reported average yearly rate for 1976–1997 of 3.4 per 1,000 births (including live births, stillbirths and fetuses from pregnancies terminated after a prenatal diagnosis of an NTD) [ 4 ]. Evidence from a number of studies has demonstrated that periconceptional use of vitamin supplements containing folic acid reduces the risk of NTDs [ 5 - 8 ]. Although the mechanism of action of this nutrient in influencing the risk of NTDs is poorly understood, the evidence of the benefit of folic acid has led many health organizations since late 1992 to recommend periconceptional folic acid supplementation, at a level of 400 μg /day for low risk women [ 9 - 11 ]. Because of concern that public education campaigns alone would not be effective in achieving optimal periconceptional folic acid intake for the majority of women, food fortification with folic acid was proposed as a strategy to ensure that all women of childbearing age increase their dietary intake of this vitamin. In November 1998, Health Canada mandated fortification of white flour and enriched pasta and cornmeal with folic acid [ 12 ]. Since diets vary, it was known that it would be virtually impossible to fortify food with folic acid at a level that ensures that the target population receives an additional 400 μg /day, while protecting the non-targeted population from an undesirably high amount. As a result, conservative levels of fortification were introduced. White flour is fortified with folic acid at a level of 0.15 mg per 100 g of flour. This intervention was expected to increase the average daily folic acid intake of women of childbearing age by about 100 μg [ 13 ]. The question of whether folic acid fortification of grain products poses any serious health risk has been controversial. The main concern has been the potential masking of vitamin B 12 deficiency, a condition that affects 10–15% of the population over age 60 years [ 14 , 15 ]. Increased folic acid intake may correct the haematologic signs of vitamin B 12 deficiency, thus delaying diagnosis and treatment of the condition while its attendant neurologic manifestations progress. Seniors may be at particular risk since the incidence of vitamin B 12 deficiency increases with age. We therefore undertook a comprehensive population based study to evaluate the effectiveness of the public health strategy of food fortification with folic acid and to determine possible adverse effects resulting from fortification. Methods Study design This evaluative study was designed as a population based study and included four components as follows: I) Determination of rates of NTDs; II) Dietary assessment; III) Blood analysis; IV) Assessment of knowledge and use of folic acid supplements. The latter three components of the study were carried out in two phases; the first phase took place prior to the introduction of mandatory fortification, from November 1997 to March 1998 and the second phase occurred from November 2000 to March 2001, after two years of implementation of mandatory fortification. This study was undertaken in Newfoundland because of the historically high rates of NTDs in the province, and because of strong interest in the health community in this initiative. Newfoundland and Labrador, with a population of approximately 500,000, has about 5,000 births annually. An urban (St. John's) and rural (Clarenville, Port Blandford, Random Island area) location in the province were chosen as the sites for this study. Data collected from these sites were compared between Phase I (November 1997 to March 1998) and Phase II (November 2000 to March 2001). Table 1 shows schematically the framework including objectives and sampling of subjects for this study. As part of this project, dietary assessment, blood analysis and assessment of knowledge and use of supplements were also carried out in a 2-phase population based study of women of reproductive age in Kingston, Ontario and environs. The results of this study will be reported elsewhere. Table 1 Framework for a two phase, multi site study to examine the effects of food fortification with folic acid Content Study objective Sample ** Location I. Rates of NTDs Determine rate of NTD-affected pregnancies, pre and post fortification Newfoundland population Newfoundland II. Dietary assessment* Determine dietary intake of folate, pre and post Fortification A) Non-pregnant women of childbearing age (19–44 years), not taking supplements containing folic acid; St. John's, Rural Newfoundland III. Blood analysis* Determine blood folate and vitamin B 12 status, pre and post fortification B) Seniors (65 years or older) not taking supplements containing folic acid or B 12 supplement and not diagnosed with anaemia. St. John's, Rural Newfoundland IV. Knowledge and intake of folic acid supplements Determine knowledge and consumption of folic acid supplements, pre and post fortification Non-pregnant women of childbearing age (19–44 years) St. John's, Rural Newfoundland * The same sample of women and seniors are analyzed in Components II and III. ** Sampling was done separately for Phases I and II. Data collection In order to examine temporal changes in the rates of NTDs in Newfoundland, data were compiled from the Newfoundland and Labrador Medical Genetics Program from 1976 to 2001. This Program ascertains cases of NTDs annually and maintains an NTD database. The database has recorded cases of NTD since 1976. Cases are identified in the following ways: provincial live birth and stillbirth notification forms, maternal-fetal medicine referrals (only one tertiary care unit in the province), and letters sent to all medical records departments of all provincial hospitals requesting data on cases assigned ICD-9/10 codes associated with NTDs or terminations for NTDs. These multiple sources are utilized to ensure complete ascertainment. NTD cases include anencephaly, spina bifida and encephalocele diagnosed in live births, stillbirths (a gestational age of 20 weeks and above or birthweight of 500 g and above) and fetuses from pregnancies terminated (at any gestational age) after a prenatal diagnosis of an NTD. For the knowledge assessment component of the study, women between the ages of 19 and 44 years were recruited through a random telephone survey. In the initial telephone survey, women were asked about their use of vitamin supplements and knowledge of the importance of folic acid for reducing the risk of NTDs or for fetal development. Women who completed the initial telephone survey were subsequently screened for their eligibility for dietary and blood assessments. Women who were not taking supplements containing folic acid and not pregnant were eligible to participate. This sampling procedure for Phase I and Phase II resulted in a response rate of 59.7% and 65.4%, respectively, with no difference between urban and rural response rates. A total of 233 women were recruited into Phase I and 204 women were recruited in Phase II, who completed components II, III and IV of the study. Seniors were recruited in the same manner as the samples of women, but were drawn only from St. John's, Newfoundland. Seniors aged 65 years or over, not diagnosed with vitamin B 12 deficiency or anaemia and not taking vitamin B 12 or supplements containing folic acid, were eligible for dietary and blood sample assessments. A total of 202 seniors were recruited in Phase I and 186 were recruited in Phase II (response rate 45.1% and 44.9%, respectively). In order to determine intakes of naturally occurring folate (the form of the vitamin found naturally in foods) pre and post fortification, and dietary intakes of folic acid (the synthetic form of the vitamin) post fortification, a Willett food frequency dietary questionnaire [ 16 ] was administered to subjects during an in-person interview. There were some modifications to the questionnaire to include common Newfoundland foods and to ensure that all foods high in folate were included. The dietary questionnaire was used to estimate an average frequency of consumption of 124 food items over the previous period of one year. The women and senior participants were also asked to provide a sample of blood in order to determine blood folate and vitamin B 12 status in Phase I and Phase II. Laboratory tests for complete blood count (CBC), red blood cell (RBC) folate, serum folate, creatinine, vitamin B 12 , plasma homocysteine (HCY) and methylmalonic acid (MMA) were conducted at the laboratories of the Health Care Corporation of St. John's. Data analysis Rate of NTDs was defined as the number of above described NTD cases, divided by the total number of live births, stillbirths, and pregnancy terminations for an NTD (termed as "births" hereafter). First we examined the temporal trend in annual rates of NTDs from 1976 to 2001 using 3-year moving average rates, then we focused on comparison of the NTD data for the most recent 11 years, identified as pre-supplementation (1991–1993), pre-fortification (1994–1997) and post-fortification (1998–2001). We regard the year 1997 as a transition period, or partial fortification period, since fortification of white flour and enriched pasta and cornmeal was permitted in Canada as of December 1996 [ 17 ]. Thus we also analysed the NTD data using 1994–1996 as a pre-fortification period. Mean daily intakes of naturally occurring folate were calculated for women aged 19–44 years and for seniors in Phase I and Phase II. Also, for Phase II, average daily intakes of folic acid from fortified foods were calculated. Data from the blood analyses were tested for normality with the Komogorov-Smirnov test, and differences between groups were tested using the non-parametric Mann-Whitney U test. The distributions of plasma MMA, plasma HCY, serum folate, RBC folate and serum vitamin B 12 were skewed. Values were therefore log transformed to give an approximate normal distribution for estimation of geometric mean and confidence intervals. Unless otherwise stated, all laboratory values presented in this paper are geometric means and 95% confidence intervals (CI). Differences in the frequency of high or low results based on reference values were tested by Pearson chi-square statistics. All data for this study were entered into SPSS (the Statistical Package for Social Sciences) Rel. 10.0 after the end of each phase. Data from the dietary interviews were analyzed using Epi-Info (Version 6.04d), while the laboratory data and the data about knowledge and use of supplements were analyzed using SPSS. Results 1. Rate of NTDs There were 617 ascertained cases of NTD among live births, stillbirths and pregnancies terminated for an NTD in Newfoundland over the 26 year period. The annual rates of NTDs in the province varied greatly over time, with the lowest rate of 2.18 per 1,000 births in 1989, and the highest rate of 5.92 per 1,000 births in 1995. The average rate of NTDs between 1976 and 1997 was 3.40 per 1,000 births. A dramatic drop is seen in 1997, in which the rate of NTDs was 2.20 per 1,000 births, down from 5.49 per 1,000 births in the previous year. The decreasing trend continued after 1998 (Figure 1 shows 3-year moving average rates). Figure 1 Rates of NTDs in Newfoundland and Labrador, 1976 to 2001 (3-year moving average rates) The rate for 1976 is a 2-yr average based on data for 1976 and 1977 and the rate for 2001 is a 2-yr average based on data for 2000 and 2001. The NTD data for the years 1991–2001 are presented in three periods in Table 2 . The mean annual rates were 4.35 per 1,000 births during 1991–1993 and 5.02 per 1,000 births during 1994–1996 (1994–96 vs 1991–93, relative risk [RR] 1.15, 95% CI 0.86–1.54, p = 0.95), and 4.37 per 1,000 births during 1994–1997 (1994–97 vs 1991–93, RR 1.01, 95% CI 0.76–1.34, p = 0.54). Table 2 Annual rates of neural tube defects (NTDs) in Newfoundland and Labrador before folic acid supplementation (1991–1993), prior to folic acid fortification (1994–1997) and after fortification (1998–2001) Period No. of cases of NTDs Total no. of births Rate per 1,000 births In live births and stillbirths In terminated pregnancies Total Pre-supplementation 1991–1993 50 40 90 20,711 4.35 Pre-fortification 1994–1997 53 50 103 23,592 4.37 Post-fortification 1998–2001 8 11 19 19,816 0.96 * The total number of births includes live births, stillbirths and terminations for an NTD. The total annual rate of NTDs fell by 78% after the implementation of folic acid fortification, from an average of 4.36 per 1,000 births during 1991–1997 to 0.96 per 1,000 births during 1998–2001 (RR 0.22, 95% CI 0.14–0.35, p < 0.0001). It is worthwhile to note that there has been no significant increase in the proportion of NTDs from terminated pregnancies since 1994. II. Dietary Assessment There was no statistically significant change in the average daily intake of naturally occurring folate among either women aged 19–44 years or seniors between Phase I and Phase II (p = 0.19 and p = 0.18, respectively). Seniors generally had dietary folate intake slightly higher than women of childbearing age. In Phase I, the average daily intake of naturally occurring folate was 306 μg/day for seniors and 262 μg/day for women aged 19–44 years, while in Phase II, the average daily intake of naturally occurring folate was 290 μg/day for seniors and 248 μg/day for women aged 19–44 years. The implementation of mandatory fortification resulted in an average additional dietary intake of 70 μg/day of folic acid in women aged 19–44, and 74 μg/day of folic acid among seniors. It is noteworthy that for the women the average daily folic acid intake due to food fortification was less than the approximately 100 μg that was previously predicted for women of childbearing age. The maximum dietary intake of folic acid due to fortification for an individual woman was 235 μg/day, and for an individual senior was 219 μg/day. III. Blood Analysis Serum folate and RBC folate increased significantly from Phase I to Phase II in both women aged 19–44 years and seniors (p < 0.001). For both age groups, there was a corresponding decrease in mean plasma HCY levels (Tables 3 and 4 ). The prevalence of low serum folate (≤6.8 nmol/L) was eliminated from the sample of seniors and the proportion of elderly participants with low stores as indicated by RBC folate levels (< 373 nmol/L) was reduced from 2.5% to 1.6%. The proportion of women aged 19–44 years with high HCY(>13.2 μmol/L) also decreased from 15.9% to 7.6% (p = 0.002) (data not shown). Table 3 Laboratory data (geometric mean and 95% confidence interval) for young women participants (age 19–44 years) in Phase I and Phase II Characteristic Phase I Phase II p value † Total participants (n) 233 204 Serum folate (nmol/L) 13.5 (12.9 – 14.1) 18.1 (17.3 – 18.9) <0.001 RBC folate (mol/L) 625 (601 – 649) 818 (784 – 854) <0.001 Plasma HCY (μmol/L) 10.2 (9.8 – 10.7) 9.2 (8.8 – 9.6) 0.001 Serum vitamin B 12 (pmol/L) 177 (169 – 186) 200 (190 – 211) 0.02 Plasma MMA (μmol/L) 0.18 (0.17 – 0.19) 0.21 (0.19 – 0.22) 0.008 † P value for the difference between Phase I and Phase II is based on a non-parametric Mann-Whitney U test. RBC denotes red blood cell, HCY denotes homocysteine, and MMA refers to methylmalonic acid. Table 4 Laboratory data (geometric mean and 95% confidence interval) for senior participants (age 65 years or over) between Phase I and Phase II Characteristic Phase I Phase II P value ‡ Total participants (n) 202 186 Serum folate (nmol/L) 14.8 (14.0 – 15.6) 23.0 (22.0 – 24.1) <0.001 RBC folate (mol/L) 745 (713 – 779) 916 (873 – 961) † <0.001 Plasma HCY (μmol/L) 13.6 (13.0 – 14.2) † 12.3 (11.7 – 12.9) † 0.001 Serum vitamin B 12 (pmol/L) 183 (173 – 194) 216 (202 – 231) <0.001 Plasma MMA (μmol/L) 0.24 (0.22 – 0.27) † 0.26 (0.24 – 0.28) 0.229 † The Komogorov-Smirnov test showed that the log transformed data were non-normal (p < 0.05). ‡ P value for the difference between Phase I and Phase II is based on a non-parametric Mann-Whitney U test. RBC denotes red blood cell, HCY denotes homocysteine and MMA refers to methylmalonic acid. There was a significant increase in mean vitamin B 12 levels in women aged 19–44 and seniors (p = 0.020 and p < 0.001, respectively, Tables 3 and 4 ). The proportion of seniors with low vitamin B 12 (<133 pmol/L) was 18.8% prior to fortification and following fortification this proportion declined to 11.8% (p = 0.032) (data not shown). A statistically significant increase in mean plasma MMA levels was observed in women subjects (p = 0.008) but not in seniors (p = 0.229) (Tables 3 and 4 ). There was also an increase in the proportion of women aged 19–44 years with MMA values above the upper reference value of 0.37 μmol/L from 3.6% in Phase I to 14.9% in Phase II (p < 0.001). There was no significant change in the proportion of abnormal MMA values in seniors. Moreover, among seniors, blood analysis showed no significant difference in mean haemoglobin concentrations, mean corpuscular volume (MCV), or proportion with abnormally high MCV (>99 fL) or low haemoglobin (<120 g/L) concentrations. IV. Knowledge and use of folic acid supplements There was a significant increase from Phase I to Phase II in the proportion of women aged 19–44 years who knew the importance of folic acid (from 33% to 46%, p < 0.001). The proportion of women taking a vitamin supplement containing folic acid increased substantially between the two time periods (from 17% to 28%, p < 0.003). Information about folic acid dosage was not collected. Discussion The results of a number of studies have led to the conclusion that periconceptional folic acid supplementation reduces the risk of NTDs [ 5 - 8 ]. Among the responses to this research evidence were calls in the early 1990s for mandatory fortification of food with folic acid. It was argued that this public health intervention would address concerns about achieving population level compliance with recommendations to women to take vitamin supplements containing folic acid before becoming pregnant and in the first weeks of pregnancy. These concerns were borne out in several Canadian studies suggesting that many caregivers [ 18 , 19 ] and women [ 20 , 21 ] remained unaware of the relationship between folic acid and NTDs. More recent studies have shown an increase in knowledge about folic acid, but supplementation rates remain low [ 22 - 25 ]. In March 1996 the US Food and Drug Administration (FDA) announced that it would permit addition of folic acid to enriched flour and other enriched cereal grain products, and that this addition would be mandatory as of January 1998. The level of fortification was set at 0.14 mg folic acid per 100 g of cereal grain product. It was determined that at this level of fortification, the intake of folate (from all sources) for the target and the general population would be kept below 1,000 μg/day, which was deemed to be the safe upper limit. This level of fortification was estimated to increase the average daily intake of folic acid in women of childbearing age by about 100 μg [ 26 ]. Subsequent to the US decision, Canada followed suit, permitting folic acid fortification at an equivalent level in December 1996 (addition of folic acid to white flour and enriched pasta and cornmeal at 0.15 mg folic acid per 100 g of flour and 0.20 mg folic acid per 100 g of pasta). In Canada, fortification became mandatory in November 1998. Rate of NTDs Our results show a highly significant drop in the rate of NTDs in Newfoundland, taking into account all identified affected pregnancies (live births, stillbirths and pregnancies terminated after a prenatal diagnosis of an NTD). The 78% (95% CI 65%–86%) reduction in the NTD rate after implementation of fortification is greater than the 18%–22% reduction predicted at current levels of fortification [ 27 , 28 ], and greater than the 19% reduction in birth prevalence of NTDs reported in the US after mandatory fortification [ 29 ]. The results in Newfoundland are closer to the 54% reduction (95% CI 34%–68%) in rate of NTDs reported in Nova Scotia after fortification [ 2 ]. De Wals et al. observed a 32% reduction (95% CI 23%–41%) in NTDs in Quebec between 1992–97 and 1998–2000 [ 30 ]. Ray et al. [ 31 ] analyzed maternal serum screening data for Ontario and observed a decline in NTD prevalence from 1.13 per 1,000 pregnancies before fortification to 0.58 per 1,000 pregnancies thereafter (prevalence ratio 0.52, 95% CI 0.40–0.67). The large reduction in the rate of NTDs in Newfoundland may be due, at least in part, to the fact that Newfoundland had higher background rates of NTDs. This population may be more sensitive to the influence of folic acid. In a large-scale public health campaign in northern and southern China, periconceptional use of 400 μg/day folic acid supplements was associated with a reduction in NTD risk of 79% for women in northern China, where the baseline NTD rate was high and similar to that observed in Newfoundland. A lower risk reduction of 41% was observed in the southern region where the pre-campaign NTD rate was much lower [ 32 ]. The 65% increase in the proportion of women taking vitamin supplements containing folic acid, from 17% in Phase I to 28% in Phase II, suggests that an increasing trend in folic acid supplementation may have played a role in the declining NTD rate in Newfoundland. In this study it was not possible to determine the individual contribution of supplementation and fortification to the trend in NTDs. The annual rate of NTDs in the pre-fortification period (1994–97) did not differ significantly from that of the pre-supplementation period (1991–93); this is true whether 1997 is excluded or included in the pre-fortification period. The increase in the rates of NTDs in 1995 and 1996 appears random and largely unexplainable. The changes in the NTD rates between 1994–1996 and 1991–1993 and between 1995–1997 and 1991–1993 were not statistically significant. In addition, our data do not show an obvious increase in the proportion of NTDs in terminated pregnancies during 1994 and 1996 (data available upon request). Dietary intakes and blood folate levels The questionnaire used in this dietary assessment was a modified Willett questionnaire [ 16 ], administered in a face-to-face interview with trained personnel. The Willett food frequency dietary questionnaire has been well validated [ 33 ] and proved easy to administer for this sample population. The daily intake of naturally occurring folate among women aged 19–44 years in this study (average 248 μg/day in the Phase II sample) was similar to values found in other studies of women's diet [ 34 , 35 ]. For seniors in Phase II, naturally occurring folate in the diet averaged 290 μg/day which was comparable to values found for persons age 49 and older in an Australian study [ 36 ]. The dietary folic acid intake due to fortification did not exceed the Tolerable Upper Intake Level (UL) of 1,000 μg folic acid/day [ 14 ] for any of the participants (this UL for folic acid does not include naturally occurring folate). It is important to note that this part of the study excluded persons taking vitamin supplements containing folic acid. While it was not possible to estimate the proportion of people in the general Newfoundland population who may be consuming more than 1,000 μg/day of folic acid from fortification and supplementation combined, it is likely that this proportion is small. The average dietary intake and maximum intake of folic acid due to fortification were 70 μg/day and 235 μg/day, respectively, for women aged 19–44 years, and 74 μg/day and 219 μg/day, respectively, for seniors. The average folic acid dose in folic acid containing over-the-counter supplements marketed in Canada is about 350 μg/day (Health Canada unpublished information). The results of this study provide strong evidence of improved blood folate status in women aged 19–44 years following mandatory fortification with folic acid. Women showed evidence of increased levels of serum and RBC folate and decreased levels of plasma HCY. These results are consistent with an earlier study examining the effect of fortification in the Framingham offspring study cohort [ 37 ]. Mandatory food fortification with folic acid has resulted in improvements in folate indices in seniors. Both mean serum folate and mean RBC folate increased following folic acid fortification (55% and 23%, respectively, Table 4 ). Consistent with this was a moderate decrease in mean plasma HCY levels among seniors by 1.3 μmol/L. Fortification of food with folic acid and an upward shift in blood folate levels is of benefit to the elderly population especially with regard to risk of cardiovascular disease. High levels of homocysteine are associated with both cerebrovascular and coronary heart disease [ 38 - 40 ]. Vitamin B 12 status There was a decline in the proportion of seniors with low vitamin B 12 levels, and there was actually a slight increase in mean vitamin B 12 levels. In vitamin B 12 deficiency, plasma MMA is usually elevated. Plasma MMA is believed to be a better indicator of vitamin B 12 status at the tissue level than serum vitamin B 12 levels are. Our study showed no change in mean MMA levels nor increased proportion of elderly with high levels. In addition, there was no change in the indicators of anaemia (i.e., haemoglobin and MCV) in seniors post fortification in our study. Thus, these results show no evidence of a deterioration in vitamin B 12 status among seniors. Furthermore, there is no evidence of improved folate status resulting in masking of the haematological manifestations of vitamin B 12 deficiency among seniors as a group. There was no evidence of deteriorating vitamin B 12 status among young women participants based on vitamin B 12 measurements. The upward trend in plasma MMA levels and higher proportion of abnormal values among young women is being further evaluated. It is unlikely that this is a direct effect of folic acid fortification and this observation is not consistent with any known effects of folic acid on vitamin B 12 status. Limitations We have documented the rate of NTDs among live births, stillbirths and terminated pregnancies known to have an NTD. It was not possible to include NTDs that may have occurred in pregnancies that resulted in a spontaneous abortion or a termination that occurred for reasons other than a congenital anomaly. This study, and other studies of fortification in Canada, are limited by the fact that there was no precise date when exposure to food fortified with folic acid began. The addition of folic acid to white flour and enriched pasta and cornmeal was permitted as of December 1996. Industry was switching to folic acid-containing enrichment premixes, especially towards the end of 1997, in anticipation of both US requirements for fortification as of January 1, 1998, and Canadian plans to implement mandatory fortification. Although this requirement did not come into force in Canada until late 1998, the Phase I (November 1997 to March 1998) subjects of our study may have consumed at least some food fortified with folic acid. This would result in an underestimate of improvements in blood folate status due to fortification, and might lead us to miss adverse effects on vitamin B 12 status. On the other hand, the fact that we observed such marked improvements in blood folate status leads us to conclude that there was a real increase in exposure to folic acid through fortification, over the study period. Another limitation of this study is the possible underestimation of folic acid intake due to fortification. Our calculations were based on the assumption that manufacturers are fortifying flour at the required level. It has been suggested that allowance for "overages" is resulting in higher amounts in the affected products [ 41 ]. Also, for enriched pasta, the required level of fortification is from a minimum of 0.20 mg/100 g pasta to a maximum of 0.27 mg/100 g. In our calculations we assumed the minimum level of fortification. We initially selected a random sample of subjects through random digit dialling, and asked eligible respondents for voluntary participation in the study. The reasonable level of response for the dietary questionnaire and blood sampling among rural and urban women aged 19–44 years suggests that with caution, we can generalize the results to all Newfoundland women of childbearing age. However, these findings may not be representative of the rest of Canada because of population differences in factors such as genetic background and dietary behaviour. These differences may also affect the generalizability of the NTD trend. The sample response rate for the dietary questionnaire and blood sampling in seniors was approximately 45% both in Phase I and in Phase II. Many of the refusals to participate were due to illness of the eligible person. Furthermore, seniors residing in long term care settings were not included. Thus our sample population of seniors may be healthier than the general population age 65 and over in the province. Conclusions The implementation of food fortification with folic acid has been accompanied by a marked decrease (78%) in the rate of NTDs in Newfoundland. The blood folate status of women aged 19–44 years improved following mandatory fortification. There is no evidence of adverse effects of the current levels of fortification on individuals aged 65 years and older. Specifically, there is no evidence to suggest an adverse effect of folic acid fortification on detection of abnormalities in vitamin B 12 status based on biochemical and haematological indices. Based on these findings, mandatory food fortification with folic acid should continue in Canada at the current levels. Over the time period of this study, the proportion of women aged 19–44 years taking a vitamin supplement containing folic acid increased. It was not possible to determine the magnitude of the separate contributions of fortification and supplementation to the decline in NTDs. Therefore, we recommend that public health efforts to promote awareness of the importance of folic acid supplementation among women of childbearing age continue. Ongoing surveillance of NTDs in Newfoundland and other parts of Canada is necessary to determine if the decline in NTD rate is maintained, and to enable further evaluation of prevention strategies. National surveillance of congenital anomalies including NTDs is a critical public health function that should be strengthened where necessary. We look forward to the results of a current epidemiologic study, funded by the Canadian Institutes of Health Research (CIHR), of NTDs in 7 Canadian provinces between 1993 and 2002. There is also research into the relationship between increased folic acid consumption and reduced risk of other congenital anomalies, cardiovascular disease and cancer [ 42 - 45 ]. As this body of knowledge grows, public health practitioners and regulators in Canada and internationally will have more evidence with which to refine existing disease prevention policies and develop new ones. Competing interests The authors declare that they have no competing interests. List of abbreviations CBC, complete blood count CI, confidence interval CIHR, Canadian Institutes of Health Research FDA, Food and Drug Administration HCY, homocysteine MCV, mean corpuscular volume MMA, methylmalonic acid NTD, neural tube defect RBC, red blood cell SPSS, Statistical Package for Social Sciences Authors' contributions SL and CM oversaw the whole study and drafted the manuscript. SL carried out the analysis of NTD rate and statistical analysis. RW, LL, KSO and HS designed the study and carried out the data collection and the dietary assessment. ER, AL and VP participated in the design and carried out the blood analysis. MC carried out the collection of NTD data. All authors read, revised and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524178.xml
544840	Homologs of the Brugia malayi diagnostic antigen BmR1 are present in other filarial parasites but induce different humoral immune responses	Background The recombinant antigen Bm R1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori . In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to Bm R1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases. In order to characterize the homologs of the Bm R1 antigen in W. bancrofti (Wb- BmR1 ), O. volvulus (Ov- Bm R1) and L. loa (Ll- Bm R1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied. Methods PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of W. bancrofti , O. volvulus and L. loa . The clones obtained were sequenced and compared to the cDNA sequence of Bm R1. Ov- Bm R1 and Ll- Bm R1 were expressed in E. coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B. malayi , W. bancrofti , O . volvulus and L. loa infections or various other parasitic infections. Bm R1, Ov- Bm R1 and Ll- Bm R1 were also tested for reactivity with the other three IgG subclasses in patients' sera. Results Wb- Bm R1 was found to be identical to Bm R1. Ov- Bm R1 and Ll- Bm R1 were found to be identical to each other and share 99.7% homology with Bm R1. The pattern of IgG4 recognition of all serum samples to Bm R1, Ov- Bm R1 and Ll- Bm R1 were identical. This included weak IgG4 reactivities demonstrated by L. loa - and O. volvulus -infected patients tested with Ov- Bm R1 and Ll- Bm R1 (or Bm R1). With respect to reactivity to other IgG subclasses, sera from O. volvulus - and L. loa -infected patients showed positive reactions (when tested with Bm R1, Ov- Bm R1 or Ll-BmR1 antigens) only with IgG1. No reactivity was observed with IgG2 or with IgG3. Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B. malayi or W. bancrofti (active infections as well as patients with chronic disease) were positive with Bm R1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses. Conclusions This study demonstrates that homologs of the Bm R1 antigen are present in W. bancrofti , O. volvulus and L. loa and that these antigens are highly conserved. Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies. We conclude that the Bm R1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis. However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.	Background Lymphatic filariasis (LF) caused by Brugia malayi and Brugia timori is endemic in several Asian countries and infects approximately 13 million people. In May 2000, The Global Program for Elimination of Lymphatic Filariasis (GPELF ) was officially formed with the goal of eliminating the disease as a public health problem by the year 2020. To this end, sensitive and specific field-applicable diagnostic tools are required for mapping the distribution of the disease and monitoring the various phases of the program. Many areas endemic for LF are remote and have poor access to well-equipped laboratories, thus a rapid and field-applicable diagnostic test is important to ensure that it can be easily be performed by field workers and reliable, reproducible, results can be obtained. For bancroftian filariasis caused by Wuchereria bancrofti , the ICT antigen card test (Binax Inc., USA ) is widely used for this purpose. This test is based on the detection of a circulating adult worm antigen of W. bancrofti . Although this antigen is also present in Brugia [ 1 ], a reliable antigen detection test for human B. malayi infection is not available. Therefore, despite its inconvenience and insensitivity, routine diagnosis of brugian filariasis is made by light microscopy of night blood. Although PCR assays are highly sensitive, these mainly detect individuals with circulating microfilariae (mf); and they are both time consuming and labour-intensive requiring well-equipped laboratory facilities. Detection of anti-filarial IgG4 antibody provides a good alternative diagnostic tool for brugian filariasis, as this antibody subclass has been shown to be elevated in active infection and decline post-treatment [ 2 - 9 ]. Recombinant antigen-based antibody assays would be preferable over assays based on parasite extracts since the former allow for unlimited supply of well-defined antigens. The Bm R1 recombinant antigen, expressed by gene pPROEXHT/ Bm17DIII (GenBank accession no. AF225296), has been shown by us to be a highly specific and sensitive antigen for IgG4 assays to detect exposure to both B. malayi and B. timori infections. The antigen has been used in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats, and evaluation in various laboratories and field trials has revealed a sensitivity of 93%–100% in detecting microfilariaemic individuals [ 9 - 13 ]. Furthermore, in some endemic areas antibodies were also detected in amicrofilaraemic individuals, indicating the sensitivity of the assay in detecting sub-patent infections in brugian filariasis [ 10 , 13 - 15 ]. The Bm R1 antigen is highly specific (99%–100%) with respect to reactivity with sera from non-filarial infections [ 11 , 12 ]. The highest prevalence of cross-reacting antibodies in other filarial infections was found in W. bancrofti , followed by Loa loa , while only one sample of nine patients with Dirofilaria infection was found to be reactive. No cross-reactivity was exhibited in patients infected with O. volvulus or Mansonella [ 11 , 16 ]. Due to its diagnostic significance, it is therefore important to characterize the BmR1 antigen more closely. The varying degree of Bm R1 recognition in other filarial infection raises the question of whether the homologous antigen is also present in W. bancrofti , L. loa and O. volvulus . In the present study we have shown that Bm R1 antigen is highly conserved (99–100% amino acid identity), and that almost identical antigens are present in the other human filarial parasites of public health importance. Interestingly, however, the ability of the hosts to mount IgG4 response to Bm R1 homologs was found to be highly variable in some infections. In addition, the antibody responses of other IgG subclasses to Bm R1 and its homolog were also investigated. Materials & methods cDNA and genomic DNA W. bancrofti microfilaria (mf), adult male and adult female cDNA libraries were obtained from the Filarial Genome Project Resource Centre (Smith College, Northampton, Massachusetts, USA genome@smith.edu ). Genomic DNA of W. bancrofti mf were prepared from samples provided by Dr. B Ravindran, Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, India . The samples were comprised of two from individuals whose serum/blood samples were negative by the Brugia Rapid test and two from individuals who were positive by the Brugia Rapid test. L. loa L3 and O. volvulus mf cDNA libraries were kindly provided by Dr. P Fischer, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany . PCR To amplify the entire Bm17DIII gene sequence the following PCR primers were used: RNF (24 mer) 5' ATT ACT GAT TAG TAT TTT ATC GTT 3' and RNR (24 mer) 5'ATG ATA AAA ATG AAT GAG AAA TAT 3'. λ phage plaques were amplified and the DNA was extracted using a λ DNA extraction kit (Qiagen, Germany ). PCR was then performed in a thermocycler (Perkin Elmer, USA ) at the following conditions: 94°C, 5 mins.; 55°C, 5 mins.; 35 cycles for 94°C, 45 sec; 55°C, 45 sec & 72°C, 90 sec; 72°C, 10 mins. Genomic DNA from W. bancrofti mf was prepared using Genispin Tissue DNA Kit (BioSynTech, Malaysia ). PCR amplifications using the above primers were then performed using the following thermocycler conditions: 94°C, 5 mins; 35 cycles for 94°C, 1 min; 55°C, 1 min & 72°C, 1 min; 72°C, 10 mins. TOPO cloning and DNA sequence analysis For sequence analysis of the gene homologs in W. bancrofti , O. volvulus and L. loa , the PCR products were cloned into TOPO-TA vector (Invitrogen, USA ), then transformed into E. coli TOP10 host (Invitrogen, USA ). The recombinant plasmids were then amplified, purified using QIAprep ® Spin Miniprep Kit (Qiagen, Germany ), and subsequently sent for sequencing (ACGT Inc, USA ). The results of the DNA sequences were analyzed using vector NTI software (Invitrogen, USA ). Subcloning, expression and purification of Ov17DIII/Ll17DIII The Bm17DIII gene homologs in O. volvulus and L. loa were subcloned into a bacterial expression vector, pPROEX-HT which contain 6-His tag (Life Technologies, USA ), then transformed into E. coli TOP 10 host cells. The recombinant bacteria were cultured in Terrific broth and placed in a shaker incubator at 37°C until the optical density reached 0.5. The culture was then induced with 1 mM IPTG (isopropyl β-D-thiogalactopyranoside) for 3 hrs at 30°C. The bacterial pellet was reconstituted with lysis buffer containing 50 mM Tris HCl (pH 8.5), 5 mM 2-mercaptoethanol and a cocktail of protease inhibitors (Roche Diagnostics, Germany ). The suspension was sonicated at 200 W for 10 minutes, followed by centrifugation at 12 000 g for 30 minutes. The resulting supernatant was purified using Ni-NTA resin (Qiagen, Germany ) and buffers containing imidazole. The protein-containing fractions were then pooled. ELISA The methodology employed was as previously reported [ 9 ]. Briefly, microtiter wells (Nunc, USA ) were coated with 100 μl of either Bm R1 (20 μg/ml) or the homologous recombinant antigens (5, 10 or 20 μg/ml) in NaHCO 3 buffer (pH 9.6). After a blocking step, serum samples (1:20 or 1:50 or 1:100) were incubated for 2 h, followed by 0.5 h incubation with the secondary antibody HRP conjugated to monoclonal anti-human IgG1 (1:6000), IgG2 (1:1000, 1:2000), IgG3 (1:1000, 1:2000) or IgG4 (1:4500) (CLB Sanquin Blood Supply Foundation, Netherlands ). Subsequently ABTS substrate (Roche Diagnostics, ) was added for 30 minutes before the optical densities (OD) were read at 410 nm with an ELISA spectrophotometer (Dynatech (now DYNEX Technologies), USA ). Serum samples were from existing serum banks, collected according to the ethical requirements of each institution. The samples were as follows: O. volvulus , L. loa , W. bancrofti , B. malayi and other parasitic infections. In addition serum samples from endemic normals (healthy and Brugia Rapid negative individuals from endemic areas in Malaysia) and non-endemic normals (healthy blood donors from Malaysia) were also tested. The O. volvulus sera were from microfilaremic from western Uganda [ 17 ]. L. loa sera were from microfilaremic individuals from the clinical department of the Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. W. bancrofti sera samples were from India; while sera from B. malayi infections, endemic normals, non-endemic normals and other parasitic infections were from Malaysia. Infections with other parasites comprised of patients from Malaysia: • whose stool specimens were positive for parasite ova/larva (single or mixed infections with Ascaris lumbricoides , Trichuris trichiura , hookworm, Strongyloides stercoralis ) • with clinical presentation and serology consistent with toxocariasis and amoebiasis • with Gnathostoma spinigerum isolated from the eye (one patient) Results Identification of the Bm R1 homolog in W. bancrofti , O. volvulus and L. loa In order to explain the pattern of antigen recognition in patients with other filarial infection we identified the homologs of the Bm R1 antigen in W. bancrofti (Wb- Bm R1), O. volvulus (Ov- Bm R1) and L. loa (LI- Bm R1). PCR of W. bancrofti cDNA libraries and W. bancrofti genomic DNA (from all 4 mf samples) produced a single band of 618 bp. PCR products were eluted from band 618 and cloned into TOPO vector. A total of 12 recombinant clones from six TOPO reactions (2 from mf cDNA, 1 from adult female cDNA, 1 from adult male cDNA and 2 from mf genomic DNA) were sequenced. A total of 31 DNA sequencing reactions were analyzed and all obtained sequences were identical. Comparison of the obtained nucleotide sequence showed that it was identical to cDNA sequence of Bm R1, irrespective whether the template DNA came from cDNA libraries, or microfilaria originated from individuals positive or negative for Brugia Rapid. For identification of the cDNA of the Ov- Bm R1 and Ll- Bm R1, a total of 5 and 3 recombinant clones were sequenced, respectively (comprising a total of 20 reactions). The Ov- Bm R1 and Ll- Bm R1 were 100% identical to each other, and only two base pairs were different from Bm R1 and Wb- Bm R1 i.e. at 97 bp and 483 bp. When the amino acid sequences were compared only one amino acid difference was observed: the uncharged polar isoleucine at position 33 was substituted by a neutral threonine (Figure 1 ). Figure 1 a) Nucleotide sequence of Bm17DIII and its homologs in W. bancrofti , O. volvulus and L. loa . Top sequence data shows DNA sequences of Bm17DIII and its homologs in W. bancrofti , O. volvulus and L. loa . b) Amino acid sequence of Bm R1 and its homologs in W. bancrofti , O. volvulus and L. loa. Bottom sequence data shows amino acid sequence of Bm R1 and its homologs in W. bancrofti (Wb- Bm R1), O. volulus (Ov- Bm R1) and L. loa (Ll- Bm R1). Comparison between Bm17DIII DNA sequence and its DNA homologs in O. volvulus and L. loa showed only two bases difference at 98 and 483. Bm R1 homologs of the amino acid sequence was identical with W. bancrofti . However, with O. volvulus and L. loa a difference occurred only at one amino acid coded by bases 97–99 i.e. a change from Ile (ATC) to Thr (ACC). Antibody reactivity to Bm R1, Ov- Bm R1 and Ll- Bm R1 recombinant antigens For IgG4-ELISA, serum samples that demonstrated average optical density (OD) readings of ≥0.300 were considered to be positive [ 9 ]. The comparison of IgG4 reactivites with Bm R1 and its homologs (Ov- Bm R1 and Ll- Bm R1) using a panel of 201 sera samples from individuals with various parasitic infections and 61 healthy controls (29 endemic normals and 32 non-endemic normals) indicated that the exchange of one amino acid had no influence on the reactivity of IgG4 antibodies. The IgG4-ELISA results showed all recombinant antigens were identical in reactivity with the various categories of sera (Table 1 ). Table 1 Comparison among IgG4 reactivities of Bm R1, Ov- Bm R1 and Ll- Bm R1 using a panel of serum samples from patients with various parasitic infections and healthy controls (endemic and non-endemic normals). Bm R1 is the antigen expressed by Bm17DIII DNA sequence; while Ov- Bm R1 and Ll- Bm R1 are the antigens expressed by the homologs of Bm17DIII DNA sequence in O. volvulus and L. loa respectively. Serum type No Positive by BmR1 (%) Positive by Ov- Bm R1 and Ll- Bm R1 (%) O. volvulus , mf positive 70 1 (1.43) 1 (1.43) L. loa , mf positive 14 6 (42.86) 6 (42.86) W. bancrofti , mf positive 33 8 (24.24) 8 (24.24) B. malayi , mf positive 28 28 (100) 28 (100) Trichuris trichiura 8 0 0 Ascaris lumbricoides 8 0 0 Mixed infection with T. trichuris , A. lumbricoides and hookworm 8 0 0 Entamoeba histolytica (invasive) 11 0 0 Toxocara 14 0 0 Gnathostoma spinigerum 1 0 0 Strongyloides stercoralis 6 0 0 Endemic normals (healthy controls) 29 0 0 Non-endemic normals (healthy controls) 32 0 0 TOTAL 262 Reactivities of Bm R1 and its homologs (Ov- Bm R1 and Ll- Bm R1) with serum antibodies of the other three IgG subclasses (IgG1, IgG2 and IgG3) using samples from O. volvulus and L. loa infected individuals showed positive reactions with only IgG1. Most IgG1 positive samples had an OD >1.000. Similarly, the reactivities of anti-filarial IgG1, IgG2 and IgG3 antibody subclasses with Bm R1 on serum samples from active and chronic cases of W. bancrofti and B. malayi showed positive reactions only with IgG1. It is also noted that sera from non-endemic normals and soil-transmitted infections also showed similar reactivities i.e. IgG1 positive and IgG2- & IgG3-negative (Table 2 ). Table 2 Results of ELISAs to detect IgG1, IgG2 and IgG3 anti-filarial antibodies in serum samples from patients with various helminthiasis and healthy controls (non-endemic normals) using Bm R1, Ov- Bm R1 and Ll- Bm R1. All antigens (tested separately) demonstrated identical results with all serum samples. Type of serum sample Number of positive results out of number of samples tested IgG1-ELISA IgG2-ELISA IgG3-ELISA O. volvulus mf+ 47/47 0/21 0/21 L. loa mf+ 14/14 0/14 0/14 W. bancrofti mf + 6/6 0/6 0/6 W. bancrofti chronic 6/6 0/6 0/6 B. malayi mf+ 10/10 0/10 0/10 B. malayi chronic 14/14 0/14 0/14 Soil-transmitted helminth infections 10/10 0/10 0/10 Non-endemic normals (healthy controls) 10/10 0/10 0/10 Discussion Bm R1, a recombinant B. malayi antigen of ~30 kDa expressed by Bm17DIII DNA coding sequence (cds), has been consistently shown to be a sensitive and specific antigen for the immunodiagnosis of brugian filariasis in studies employing either ELISA or immunochromatographic rapid test (Brugia Rapid) formats [ 9 , 11 - 13 , 15 ]. When compared with the DNA sequences in GenBank, Bm17DIII cds exhibited 94% homology with the reported EST sequence derived from B. malayi microfilaria cDNA (GenBank AW244981). Southern blot hybridization assays performed on cDNA libraries of L3, L4, mf, adult male and adult female B. malayi showed that it is present in all of the above stages (Rahmah et al ., unpublished data). Bands of the correct molecular weight were observed in a Western blot of B. malayi mf, adult male and adult female soluble antigens probed with monopurified antibody to Bm R1 (Rahmah et al., unpublished data). Multicenter evaluations performed with Brugia Rapid showed variable reactivity of Bm R1 to sera of W. bancrofti -infected patients. Reactivity to sera from Chennai, India was 54.5% (12/22); from Indonesia was 70% (14/20) and from the Cook Islands was 90% (9/10) [ 12 , 15 ]. The wide variation in the reactivity of the assay in Bancroftian filariasis in the above three geographical areas prompted us to undertake the current investigation. The present study has shown that the homolog in W. bancrofti is identical to the cDNA of Bm R1 – irrespective of the source of the parasites – whether the mf were isolated from the individuals whose sera showed positive or negative reactivity with the Brugia Rapid test. Thus the observed differences in the reactivity of Bm R1 antigen with W. bancrofti sera collected from different geographical regions does not appear to be due to genotypic variability between different isolates of mf. Further studies are currently underway to determine if the variability in the expression of the gene could account for the variability in the Brugia Rapid results with serum samples collected from W. bancrofti infected individuals. PCR experiments were performed on the W. bancrofti genomic DNA samples to obtain an amplicon with a size greater than 618 bp (since an intron is expected to be present in genomic material). However, only one prominent band of 618 bp was obtained (very occasionally a faint band of >1 kb was observed which was shown later to be due to unspecific amplification). PCR on W. bancrofti genomic DNA to amplify the intron sequence (using primers based on the Bm17DIII intron) produced a sequence that shared ~75% homology to the intron of Bm17DIII . This is believed to be an amplification on another part of W. bancrofti genome, since PCR using a pair of internal primers that flank the possible intron site produced a PCR product of ~300 bp (a size that is expected if there was no intron). Conversely, amplification of B. malayi genomic material produced two kinds of amplicons: 618 bp and 1010 bp. The latter was comprised of an intron (393 bp) and two flanking exons (237 bp and 381 bp), the sequences of which were consistent with B. malayi data at TIGR website . Thus at Universiti Sains Malaysia, genomic DNA of Wb17DIII was found to be intronless, whereas genomic DNA of Bm 17DIII was shown to have two variants (i.e one with and one without an intron). These results, though seemingly controversial, were a result of exhaustive efforts with appropriate PCR controls. Data from other laboratories will hopefully confirm these results. Anti- Bm R1 IgG4 was detected in 84.6% (44/52) of L. loa sera but generally not detected in O. volvulus serum samples [ 11 , 16 ]. Ov- Bm R1 and Ll- Bm R1 were identical to each other and 99.7% similar to Bm R1 (and to Wb- Bm R1) on the nucleotide level (Figure 1 ). Ov- Bm R1 and Ll- Bm R1 were found to display identical reactivity compared to Bm R1 when tested with IgG4-ELISA on a panel of serum samples (Tables 1 & 2 ). Therefore, the difference of one amino acid between Bm R1 and its homologs (Ov- Bm R1 and Ll- Bm R1) did not alter their antigenicity. It is interesting to note that although IgG4 has been shown to be elevated in onchocerciasis with assays using other recombinant antigens [ 18 , 19 ], the IgG4 reactivity to Bm R1 or Ov- Bm R1 in O. volvulus was generally negative. One possible explanation for this is that adult worms mostly express Ov-BmR1 and the immune response to O. volvulus is predominantly due to mf [ 20 ]. This may explain the very poor IgG4 response to Bm R1 and Ov- Bm R1. It is possible that the uptake of antigen from lymphatic filariae by antigen presenting cells is significantly different compared to O. volvulus (where adult worms and mf reside either in sub-dermal nodules or in the skin). The Bm R1, Ov- Bm R1 and Ll- Bm R1 recombinant antigens were also used to determine if IgG1, IgG2 or IgG3 antibodies in O. volvulus and L. loa serum samples were reactive with the recombinant proteins. In addition, the three IgG subclasses were also tested with Bm R1 on assays using sera collected from patients with B. malayi and W. bancrofti infections. In all cases only anti-filarial IgG1 was reactive, while anti-filarial IgG2 and IgG3 assays were consistently negative. It is important to note that IgG1 antibodies to Bm R1 and its homologs are unspecific and without any diagnostic value. The Bm R1 antigen obviously contains widespread epitopes that are recognized by IgG1 antibodies. Thus based on the current study, Bm R1 and its homologs in W. bancrofti , O. volvulus and L. loa induce IgG antibody responses restricted to IgG1 and IgG 4 subclasses only. Unlike the anti-filarial IgG4 response in B. malayi infection, the IgG4 response to Bm R1 in W. bancrofti and L. loa was not consistently detected in all infected individuals, indicating that this recombinant antigen will not be of much use in the diagnosis of these two filarial infections. Although IgG1 response to Bm R1 was observed in all the filarial infections tested, it lacks specificity since it was also positive when tested with serum samples from normal individuals and with those infected with other parasites. Conclusions The study demonstrates the presence of identical and almost identical homologs of the diagnostic Bm R1 antigen in other filarial parasites. However, they do not seem to induce consistent antibody responses in all infected subjects. Thus the immunogenicity of Bm R1 in brugian filariasis appears to be clearly different from that of bancroftian filariasis, onchocerciasis and loiasis. Competing interests The author(s) declare that they have no competing interests. Authors' contributions RN – was the principle researcher, designed the study, supervised the experiments and result analysis, wrote the first draft of the manuscript. RAAA – performed the experiments and participated in the analysis of the data. BR – provided parasite materials, collected patients' sera, edited the paper.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544840.xml
534800	Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins	Since thousands of years humans have utilized insect silks for their own benefit and comfort. The most famous example is the use of reeled silkworm silk from Bombyx mori to produce textiles. In contrast, despite the more promising properties of their silk, spiders have not been domesticated for large-scale or even industrial applications, since farming the spiders is not commercially viable due to their highly territorial and cannibalistic nature. Before spider silks can be copied or mimicked, not only the sequence of the underlying proteins but also their functions have to be resolved. Several attempts to recombinantly produce spider silks or spider silk mimics in various expression hosts have been reported previously. A new protein engineering approach, which combines synthetic repetitive silk sequences with authentic silk domains, reveals proteins that closely resemble silk proteins and that can be produced at high yields, which provides a basis for cost-efficient large scale production of spider silk-like proteins.	Review Types of spider silk Spiders and silks always go together. Currently there are over 34,000 described species of spiders, all of which have a varied tool kit of task-specific silks with divergent mechanical properties [ 1 - 6 ]. Although some spiders may use silk sparingly, most make rather elaborate nests, traps and cocoons typically using more than one type of silk (Figure 1 ), which are produced by a wide and diverse range of glands, ducts and spigots. Among the various spider silks the major ampullate (MA) silk, which forms the primary dragline, is extremely tough. MA silk reveals a tensile strength that is comparable to Kevlar (4 × 10 9 N/m 2 ) coupled with a reasonable viscoelasticity (dragline 35 %, Kevlar 5 %). Spiders use dragline silk as a strong yet flexible structural element in the web, providing a framework to which other silks are attached, and as a life line when a spider is dropping off to escape an enemy. Minor ampullate (MI) silk, used for structural reinforcement in construction of the web, has a similar high tensile strength in comparison to major ampullate silk but has little elasticity [ 7 , 8 ]. Due to the low elasticity of MI silk it is irreversibly deforming when stretched. An orb web's capture spiral, in part composed of viscid silk formed by the flagelliform gland, which is therefore named flagelliform silk, is stretchy and can triple in length before breaking, but provides only half the tensile strength of major ampullate silk [ 9 ]. The combination of strength and stretchiness gives the capture spiral a toughness (energy to break) greater than elastin, tendon, silkworm silk, bone, synthetic rubber, Kevlar, and high-tensile steel. Composition and structural architecture of spider silks Spider silks are protein polymers that display extraordinary physical properties [ 1 - 4 , 8 ], but there is only limited information on the composition of the various silks produced by different spiders. Among the different types of spider silks, draglines from the golden orb weaver Nephila clavipes and the garden cross spider Araneus diadematus are most intensely studied. Dragline silks are generally composed of two major proteins [ 5 , 10 - 13 ] and it remains unclear whether additional proteins play a significant role in silk assembly and the final silk structure. The two major protein components of draglines from Nephila clavipes are termed MaSp1 and MaSp2 ( M ajor a mpullate S pidroins) and from Araneus diadematus ADF-3 and ADF-4 ( A raneus D iadematus F ibroin). The dragline silk proteins have apparent molecular masses between 180 kDa and 720 kDa depending on the conditions of analysis [ 14 - 16 ]. It is assumed that, based on amino acid composition, within the dragline fiber the molecular ratio between MaSp1 and MaSp2 and between ADF-4 and ADF-3 is approximately 3 to 2 [ 10 , 11 , 17 ]. Based on DNA analysis it could be shown that all spider silk proteins are chains of iterated peptide motifs (so called repeating units) (Figure 2 ). The small peptide motifs can be grouped into four major categories: GPGXX (with X often representing Q), alanine-rich stretches (A n or (GA) n ), GGX, and spacers (Figure 2A ). A fifth category is represented by non-repetitive (NR) regions at the amino- and carboxyl termini of the proteins (Figure 2 ), often representing polypeptide chains of 100 amino acids and more [ 7 , 10 , 11 , 18 - 22 ]. So far the largest sequence information could be obtained for flagelliform silk from Nephila clavipes (Figure 2B ). This flagelliform silk protein is translated from a ~15.5 kb mRNA transcript originating from a 30 kb Flag locus [ 9 , 23 ]. The coding sequence is divided into 13 exons. The NR amino-terminal region is split between exons 1 and 2. All of the other exons are found to encode exactly one repeating unit, built from the described motifs (Figure 2B ). The final exon 13 in addition includes the NR carboxyl-terminal region. On the basis of several studies, the major categories of peptide motifs in spider silk proteins have been assigned structural roles [ 24 - 28 ]. The GPGXX motif has been suggested to be involved in a β-turn spiral, probably providing elasticity, based on structures of comparable proteins [ 29 - 32 ]. If elasticity is due to GPGXX β-spirals, then this motif should be found in the more elastic silks. Flagelliform silks, which show the highest elasticity with more than 200 %, consist of contiguous repeats of this motif for at least 43 times in each repeating unit (Figure 2B ). The only non-flagelliform silk proteins with GPGXX motifs are MA proteins MaSp2, ADF-3, and ADF-4, which also display some viscoelasticity. In accordance to the lower elasticity of dragline silk in comparison to flagelliform silk the number of tandemly arrayed repeats depicts at most 9 concatenated GPGXX motifs before interruption by another motif [ 1 , 21 ]. Alanine-rich motifs contain typically 6–9 alanine residues and have been found to form crystalline β-sheet stacks leading to tensile strength [ 6 , 24 , 25 , 12 ]. The MA and MI silks are both very strong, and at least one protein in each silk (there are always pairs) contains the A n or (GA) n motif. Interestingly, this motif is not found in flagelliform silks. A glycine-rich 3 1 -helix is adopted by the GGX motif forming an amorphous matrix that connects crystalline regions and that provides elasticity [ 26 , 33 , 34 ]. The postulated GGX motif is widely distributed and this motif can be found in MA, MI and flagelliform silks (Figure 2A ). Several groups have suggested that the motifs GPGXX and GGX might be involved in forming an amorphous matrix, which would provide the elasticity of the fiber. The spacers contain charged groups and separate the iterated peptide motifs into clusters. Non-repetitive termini are common to all sequenced MA, MI and flagelliform silks belonging to the Araneoidea family with highly conserved carboxyl-terminal sequences [ 19 , 35 , 36 ]. The structural impact of the spacer and terminal regions is so far undetermined [ 37 ]. Recent findings on single NR-regions of ADF-3 and ADF-4 (without additional repeating units) revealed a secondary structure comprising α-helices as determined by Circular Dichroism and they seem to retain this structural feature in proteins that additionally contain repeating units [ 36 ]. It can be speculated that the α-helical NR carboxyl-termini might play a crucial role during assembly of the silk fiber [ 19 , 36 , 38 ]. Natural spider silk assembly Silk assembly in vivo is a remarkable process. For instance, dragline silk proteins are stored at concentrations up to 50 % (w/v) in the respective glands [ 39 ]. This highly concentrated protein solution forms the silk dope (spinning solution), which displays properties of a liquid crystal [ 40 - 42 ]. Therein, the polyalanine motifs are thought to adopt an α-helical conformation, while the glycine-rich motifs form either β-turns or random coil conformation [ 39 , 43 , 44 ]. Thread assembly is initiated during a passage of the silk dope through the spinning duct accompanied by extraction of water, sodium and chloride [ 45 , 46 ]. Simultaneous secretion of potassium and hydrogen ions into the lumen of the duct lowering the pH from 6.9 to 6.3 is thought to initiate partly unfolding of the proteins by disrupting their water shell and altering coulombic forces [ 42 , 45 - 48 ]. The silk proteins are thought to extend somewhat, align and get packed much closer in the extensional flow-field of the draw-down taper found in the distal part of the duct. As the hydrophobic polyalanine segments of the silk proteins align and are drawn closer together by extensional flow, they are exposed to an increasingly hydrophobic environment, which might trigger their conversion from an α-helical to a β-pleated structure resulting in the formation of numerous interchain hydrogen bonds. The latter would act as multifunctional crosslinks at nodes between the more mobile glycine-rich segments. Thus the assembly of the thread can be seen as a liquid-crystalline phase transition involving separation into polymer-rich and solvent-rich phases [ 47 ]. While some aspects of spider silk assembly have been unraveled, the contribution of the individual silk proteins to the assembly process still needs to be resolved in more detail. Comparative studies of the two major dragline silk proteins of Araneus diadematus , ADF-3 and ADF-4, revealed that, although their amino acid sequences are rather similar [ 5 ], they display remarkably different solubility and assembly characteristics: While ADF-3 is soluble even at high concentrations [ 49 ], ADF-4 is virtually insoluble and self-assembles into filamentous structures under specific conditions [ 50 ]. At a closer look, the different solubilities of ADF-3 and ADF-4 could be explained by the hydrophobicities of the two proteins. The hydrophilic ADF-3 interacts favourably with the aqueous solvent and thus remains soluble under most conditions. In contrast, the more hydrophobic ADF-4 favours interactions with other protein molecules and thus tends to aggregate. Interestingly, all pairs of dragline silk proteins from different spider species display a common distinct distribution of hydrophobicity. In direct comparison, MaSp1 / ADF-4 proteins generally display relatively high hydrophobicity, while the corresponding MaSp2 / ADF-3 partner protein is more hydrophilic [ 50 ]. Applications for spider silks Laboratory-scale production of spider silk would initiate a new generation of ecological materials. Spider silk is for instance a promising tool with broad usability in medical devices. In the middle ages spider webs were used as wound dressing – some reports are even dated back to ancient Greek and Roman cultures. Silkworm silk does not cause allergic reactions and it is thought that spider silk behaves similarly [ 51 ]. The unmatched toughness of spider silk would allow to improve several medical products such as wound closure systems, band-aids, and extremely thin sutures for neurosurgery. Additionally, spider silks can be further used in artificial ligaments and tendons for durable implants. High performance fibers built from spider silks can be employed in several technical and industrial applications. In addition to specialty ropes and fishing nets, spider silk can be utilized for parachutes, ballistic applications (body armor), sporting goods, textiles, and lightweight constructions for airplanes [ 52 , 53 ]. Therefore, one day industrially produced spider silk could out-compete man-made fibers. Production of recombinant spider silk proteins Recombinant production of spider silk proteins has been complicated by the highly repetitive nature of the underlying genes, by their high gc -content, by the length of the constructs, and by the specific codon usage of spiders. In first studies, in vitro translation of mRNA from excised major ampullate glands of Nephila clavipes was performed using tRNA from E. coli , but translation was discontinuous [ 14 , 54 ]. In the era of recombinant proteins and genetic engineering one would envisage to easily produce spider silk proteins (mainly from draglines) in microbes or cell culture. Unfortunately, no dragline silk gene has been cloned in its entirety and only sequence data from the 3' end of partial cDNA clones of dragline genes from Nephila clavipes and Araneus diadematus and other spiders have been reported [ 10 , 11 , 20 - 22 ]. Therefore, all recent studies used partial cDNA constructs of dragline silk genes to produce recombinant silk proteins in E. coli [ 55 ], in MAC-T (bovine) and BHK (hamster) cells [ 49 ], or in insect cell lines from Spodoptera frugiperda using the baculovirus expression system [ 50 ]. The most promising expression system seems to be the baculovirus system, since it was possible to efficiently produce dragline silk components at a high yield. Designing of synthetic spider silk proteins Cloning strategies for designing genes for bacterial, yeast or plant expression have been developed to produce recombinant silk-like proteins closely resembling natural dragline [ 29 , 36 , 56 - 62 ] or flagelliform silk proteins [ 63 ]. Since gene manipulation and amplification of spider silks is difficult by PCR due to the repetitive nature of silk genes, cloning strategies involved engineering of synthetic DNA modules. These modules were optimized for the codon usage adapted by the corresponding expression host. The use of synthetic modules constructed from small size oligonucleotides repeats has allowed control over primary gene and protein sequence and final protein size. Tobacco, potatoes, the yeast Pichia pastoris and mainly E. coli have been utilized as expression hosts for synthetic genes yielding proteins with up to 150 kDa [ 29 , 56 - 62 ]. Unfortunately, expression levels from the synthetic genes have been low and mostly the recombinant silk proteins represented only up to 5% of the total protein in the cell [ 13 ]. Although once production levels of up to 1000 mg/l of cell culture have been reported [ 57 ], large losses in yield are encountered during purification due to precipitation and non-specific interactions. For the microbial expression systems, yields of purified proteins have been generally in the 10 to 40 mg/l range (>90% purity) [[ 55 , 56 , 59 , 60 ], summarized in [ 13 ]]. In a recent study, genes coding for spider silk-like proteins were generated using a cloning strategy, which was based on a combination of synthetic DNA modules and PCR-amplified authentic gene sequences (Figure 3 ) [ 36 ]. This approach was in contrast to previous protein designs, which did not include the carboxyl-terminal NR-regions that are found in all dragline silks. The dragline silk proteins ADF-3 and ADF-4 [ 10 ] from the garden spider Araneus diadematus were chosen as templates for the synthetic constructs. A seamless cloning strategy [ 64 ] allowed controlled combination of different synthetic DNA modules as well as authentic gene fragments. A cloning vector was designed comprising a cloning cassette with a spacer acting as placeholder for synthetic genes [ 36 ] (Figure 3B ). To mimic the repetitive sequence of ADF-3, two modules have been designed. The sequence of one module, termed A, was derived from the poly-alanine containing consensus sequence of ADF-3 (Figure 3A ). The sequence of a second module termed Q contained four repeats of the GPGQQ motif. In a first cloning step the spacer region of the cloning vector was replaced by one of the synthesized DNA modules. Subsequently two modules could be joined in a site-directed way. To study different length repeat units, one or two Q modules were combined with one A module to obtain (AQ) (Figure 3B ) or (QAQ) (Figure 3C ). The complementary 3'-single strand extensions gg (sense) and cc (antisense) were used for connecting two modules (Figure 3B ). Thus the DNA sequence required to link two modules was confined to a glycine codon ( ggn ). Glycine is naturally abundant in spider silk proteins (~30%), therefore modules could be designed to match authentic amino acid sequences. Since the arrangement of the cloning cassette's elements remained unchanged upon cloning, repeat units could be multimerized to generate synthetic genes coding for the repetitive proteins (rep-proteins) (AQ) 12 and (QAQ) 8 (Figure 3C ). The repetitive part of ADF-4 is generally composed of a single conserved repeat unit displaying only slight variations. These variations were combined and one consensus module termed C has been designed (Figure 3A ), which was multimerized to obtain the rep-protein C 16 (Figure 3C ). ADF-3 and ADF-4 both display NR-regions at their carboxyl termini, comprising 124 and 109 amino acids respectively. Gene sequences coding for these regions were amplified by PCR, and codons problematic for bacterial expression were changed to more suitable codons by site directed mutagenesis. In the described system, all synthetic genes could be combined with the appropriate authentic NR-regions. Additionally NR3 and NR4 could be expressed individually. All constructs could be purified by a heat step followed by an ammonium sulfate precipitation [ 36 ], which has been employed in previous studied for purifying spider silk proteins [ 35 , 62 ]. Based on this protein engineering approach, which combines synthetic repetitive sequences with authentic NR-regions, proteins closely resembling authentic silk proteins could be produced at high yields. Bacterial production in Erlenmeyer flasks yielded similar protein amounts for all constructs. Yields of individual preparations ranged from 10 to 30 mg of purified protein per liter of culture medium. Fermentation of cells increased the yield of purified protein to 140 and 360 mg/l (purity >95%). Therefore, the established bacterial expression system provides the basis for cost-efficient large scale production of spider silk-like proteins. Assembly of recombinant spider silks One important feature of spider silk proteins is their storage at high protein concentrations (up to 50% w/v) in the dope without apparent aggregation or assembly. However, spider silk proteins can rapidly assemble into highly stable fibers when needed. The determination of solubility and self-assembly of recombinant spider silk proteins is therefore important to create commercially available silk fibers. For instance, pH-shifts are involved in natural silk assembly, but the exact function of acidification during spider silk assembly has not yet been determined. It seems likely that negatively charged groups are protonated reducing the net charge of spider silk proteins. Phosphoryl groups of phosphorylated amino acid residues, which have been detected in dragline silk [ 65 ], display pK A -values [ 66 ] near the range of the pH-shift observed during the spinning process and thus could be involved in triggering silk assembly. Therefore, changes in pH can be used to initiate assembly of recombinant spider silk proteins [ 36 ]. Interestingly the intracellular pH 6.3 of Sf9 cells (derived from the fall armyworm Spodoptera frugiperda ) used for producing ADF-4 corresponds to the pH in the spinning dope prior to silk thread assembly [ 50 ]. These pH conditions, among additional factors, might be involved in initiating aggregation of ADF-4 within the cytosol of Sf9 cells [ 50 ]. Surprisingly, investigating the aggregates in adf-4 expressing cells revealed filaments that coiled throughout the cytoplasm, whereby most of the cells contained only one or few filaments. In contrast, Sf9 cells infected with control viruses or the adf-3 encoding virus never produced such filaments. The diameter of the ADF-4 filaments (0.2 – 1.5 μm) was in the range of native dragline silks (1.0 – 4.0 μm), but length of the filaments formed in the Sf9 cells seemed to be constrained by the volume of the cells, making them too short for mechanical force measurements. Strikingly, the purified ADF-4 filaments (Figure 4B ) showed a similar morphology and chemical stability in comparison to natural dragline silk threads of Araneus diadematus [ 50 ]. Phosphate, like other lyotropic ions, is known to increase the surface tension of water, promoting hydrophobic interactions [ 67 ]. In the case of spider silk proteins it is likely that the addition of phosphate initiates interactions between the hydrophobic poly-alanine motifs, causing the aggregation of the proteins. Accordingly aggregation of polyalanine-rich proteins is pronounced in comparison to synthetic silks which contained one third less poly-alanine motifs [ 36 ]. Strikingly, recombinant spider silk proteins are highly soluble in most aqueous solutions, but form nanometer-sized fibers upon addition of methanol, phosphate or other suitable ions (Figure 4A ). Artificial spinning of spider silks A remaining critical step concerning commercial production of silk fibers is the successful spinning of recombinant proteins into fibers resembling the natural silks in their microstructure and in their mechanical properties, which are outstanding by any measure. Besides the chemical parameters discussed above, several mechanical parameters play important roles in generating silk. To draw silk under natural spinning conditions, spiders attach their dragline to an object with glue from the piriform glands, before drawing the silk out by moving away or by descending and using their weight to draw the silk. It is common practice to take advantage of the drawing process by the forced silking of captive animals to collect silk for experiments. Analysis of the differences between naturally and forcibly spun dragline silk provided evidence for discrepancies in their material properties [ 44 , 68 , 69 ]. Forced spinning under spinning speeds ranging from 0.1 to 400 mm/s and temperatures ranging from 5 to 40°C revealed dramatic differences in strain at breaking, breaking energy, initial Young's modulus and point of yielding [ 70 ]. Therefore, in case of spinning recombinant silk proteins in vitro several aspects have to be taken into account to gain materials with expected properties. Several attempts are reported in the literature and even more have been performed to wet-spin recombinant spider silk proteins. In a first attempt, microfabricated spinnerets were constructed using silicon microfabrication methods [ 71 - 73 ]. These spinnerets allowed for the production of meters of silk fibers from solutions containing as little as 10 mg of protein. First the spinneret was validated and tested by producing fibers from dissolved silk from the silkworm Bombyx mori [ 71 ], before solubilized dragline silk from Nephila clavipes was wet-spun [ 72 ]. The diameters and mechanical properties of the regenerated silkworm silks converged the native silk ones. However, the wet-spun spider silks exhibited diameters of about 40 μm compared to the natural fiber diameter of 2.5 to 4.0 μm with mechanical properties that did not match the natural ones [ 72 ]. Other attempts of wet-spinning revealed fiber diameters of approximately 10 – 60 μm [ 49 , 74 ]. These fibers were subjected to either single or double postspinning draw, first in 70 to 80 % methanol (single and double draw) and then in water (double draw only) to increase their mechanical properties. Fibers subjected to higher draw ratios displayed greater toughness, tenacity, and modulus values [ 49 ]. However, even the best values obtained by such technique were in the range of the regenerated Nephila fibers [ 72 ], but lower than the reported values for natural dragline silks [ 2 ]. Perspectives Engineering of precision fibers The future objective might not be to prepare identical copies of natural silk fibers, but rather to capture its key structural and functional features in designs that could be useful for engineering applications (Figure 5 ). Using "protein engineering" based on knowledge achieved from investigations of the natural silks, artificial proteins can be designed that allow bacterial synthesis at high yields [ 75 ]. The soluble synthetic silk would be able to assemble into protein fibers with desired properties, which includes the possibility to specifically functionalize the fiber surface by chemical cross-linking with biologically or chemically active groups. Such protein fibers could be optimized by additional protein engineering in order to gain fibers that allow the formation of interconnected nano- or micrometer-scale networks, which are capable of various biological, chemical or physical processes (e.g. enzymatic reactions, chemical catalysis, electronic signal propagation, etc.) (Figure 5 ). The main strategy would be to modify the monomeric proteins either with the desired functionality prior to assembly into fibers, or to incorporate a reactive group that will subsequently permit the conjunction with desired functionality after the fibers have assembled. Protein fibers could for instance be covalently linked with external units through chemically specific amino acid side chains (e.g. SH-groups of cysteines) [ 76 - 78 ]. Since the physical and chemical properties of bio-polymers and their assembly processes depend on the amino acid composition of the underlying polypeptide, engineering "synthetic" proteins with specific structural features will create a new class of fibrous proteins. However, to design new biomaterials based on spider silk, all properties of the underlying proteins have to be analyzed and in the best case successfully mimicked [ 35 ]. Therefore, the crucial design features of both the feedstock of the dope and the spinning process have to be closely adopted, which would allow for managing the commercial production of new materials.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC534800.xml
548263	Activation of α1A-adrenergic receptor promotes differentiation of rat-1 fibroblasts to a smooth muscle-like phenotype	Background Fibroblasts, as connective tissue cells, are able to transform into another cell type including smooth muscle cells. α 1A -adrenergic receptor (α 1A -AR) stimulation in rat-1 fibroblasts is coupled to cAMP production. However, the significance of an increase in cAMP produced by α 1A -AR stimulation on proliferation, hypertrophy and differentiation in these cells is not known. Results Activation of the α 1A -AR in rat-1 fibroblasts by phenylephrine (PE) inhibited DNA synthesis by 67% and blocked the re-entry of 81% of the cells into S phase of the cell cycle. This cell cycle blockage was associated with hypertrophy characterized by an increase in protein synthesis (64%) and cell size. Elevation of cAMP levels decreased both DNA and protein synthesis. Inhibition of adenylyl cyclase or protein kinase A reversed the antiproliferative effect of cAMP analogs but not PE; the hypertrophic effect of PE was also not altered. The functional response of rat-1 cells to PE was accompanied by increased expression of cyclin-dependent kinase (Cdk) inhibitors p27 kip1 and p21 cip1/waf1 , which function as negative regulators of the cell cycle. Stimulation of α 1A -AR also upregulated the cell cycle regulatory proteins pRb, cyclin D1, Cdk 2, Cdk 4, and proliferating cell nuclear antigen. The antiproliferative effect of PE was blocked by p27 kip1 antisense but not sense oligonucleotide. PE also promoted expression of smooth muscle cell differentiation markers (smooth muscle alpha actin, caldesmon, and myosin heavy chain) as well as the muscle development marker MyoD. Conclusions Stimulation of α 1A -AR promotes cell cycle arrest, hypertrophy and differentiation of rat-1 fibroblasts into smooth muscle-like cells and expression of negative cell cycle regulators by a mechanism independent of the cAMP/PKA signaling pathway.	Background Alpha1-adrenergic receptors (α 1 -ARs) are members of the G-protein-coupled receptor superfamily. Both pharmacological and molecular cloning studies have indicated the existence of multiple subtypes of α 1 -ARs including α 1A/C -AR, α 1B -AR, and α 1D -AR [ 1 - 4 ]. α 1 -ARs play a key role in a variety of physiological processes, such as contraction of vascular and cardiac muscle, contraction of the spleen, liver glycogenesis, or melatonin secretion in the pineal gland [ 3 , 4 ]. Activation of α 1A -AR promotes hypertrophy of cardiac myocytes [ 5 , 6 ]. Recently it has been shown that all three subtypes of α 1 -AR are also expressed in rat aortic adventitial fibroblasts and vascular smooth muscle cells (SMC) [ 7 ] and their activation with norepinephrine stimulates migration, proliferation and protein synthesis [ 8 , 9 ]. However, norepinephrine increased SMC hypertrophy, but not DNA synthesis, through α 1A -AR stimulation in uninjured aorta whereas norepinephrine stimulated proliferation of adventitial fibroblasts through the α 1B -AR subtype [ 8 ]. Nonvascular fibroblasts, including cardiac fibroblasts [ 7 , 10 ], generally do not express α 1 -AR and have been used for stable transfection of different subtypes of α 1 -AR to study their respective functions. However, a recent study showed the expression of a functional α 1A -AR in primary tendon fibroblasts [ 11 ]. In rat-1 cells, a transformed cell line from embryonic fibroblast, expressing different subtypes of α 1 -AR, phenylephrine (PE), an α 1 -AR agonist, activates phospholipase D and releases arachidonic acid [ 12 ]. However, unlike SMC, activation of α 1 -ARs in rat-1 cells also increases cAMP levels and PKA activity [ 12 ]. α 1A -AR is more efficiently coupled to phospholipase D activation, arachidonic acid release and cAMP than α 1B -AR or α 1D -AR in these cells [ 12 ]. Activation of α 1A -AR expressed in COS-7 and HeLa cells [ 13 ] and α 1B -AR or α 1D -AR in COS and CHO cells [ 14 ] also increase cAMP levels. In HepG2 and M12 cells expressing α 1B -AR, PE causes cell scattering and inhibits proliferation through activation of MAP kinases [ 15 ]. The family of connective tissue cells includes fibroblasts, cartilage cells, bone cells, fat cells and smooth muscle cells. Fibroblasts seem to be able to transform into any of other members of the family – in some cases reversibly – although it is not clear whether this is a property of a single type of fibroblast that is pluripotent or of a mixture of distinct types of fibroblasts with more restricted potentials. These transformations of connective tissue cell type are regulated by the composition of the surrounding extracellular matrix, by cell shape, and by hormones and growth factors [ 16 ]. Heterologous expression of α 1A -ARs in CHO cells inhibits basal and growth factor-stimulated DNA synthesis, in contrast to the α 1D -AR [ 17 ]. A recent study in the same model has reported cAMP as the mediator of the antiproliferative effect of α 1A -AR stimulation [ 18 ]. Therefore, it is possible that activation of α 1A -AR with PE in rat-1 cells affects their growth and/or differentiation status. To test this hypothesis, we have investigated the effect of PE and cAMP modulators on proliferation, growth and morphology in rat-1 cells expressing α 1A -ARs. Moreover, we have examined the effect of PE and cAMP modulators on the expression of cell cycle regulators and muscle cell markers, because of the ability of fibroblasts to differentiate into myofibroblasts. Our results show that activation of α 1A -ARs in rat-1 cells exerts profound effects promoting hypertrophy and expression of specific smooth muscle cell markers. We also show here that α 1A -AR-induced cessation of DNA synthesis is independent of cAMP and involves the expression of cyclin-dependent kinase (Cdk) inhibitor, p27 kip1 . Results Stimulation of α 1A -AR inhibits DNA synthesis at the G 1 /S checkpoint of the cell cycle in rat-1 fibroblasts Rat-1 fibroblasts stably transfected with α 1A -AR expressed 288 ± 2 fmol/mg protein of receptors [ 12 ]. Cells at 80% confluency were serum-deprived for 48 h in DMEM and then incubated with PE (2, 5 and 10 μM) for different periods prior to the addition of [ 3 H]thymidine. PE decreased basal [ 3 H]thymidine incorporation in a concentration-dependent manner with a maximum effect at 10 μM PE (Fig 1A ). Reduction of basal DNA synthesis was significant after 12 h of treatment with a maximum effect at 18 h of incubation with PE (> 65% decrease) (Fig 1B ). Rat-1 fibroblasts stably transfected with α 1A -AR incorporate [ 3 H]thymidine in DNA in a time-dependent manner (from 6 to 24 h), even after 48 h of serum-deprivation (Fig 1B ). Figure 1 Effect of PE on [ 3 H]thymidine incorporation in rat-1 cells. Cells were grown in 24-well plates in DMEM containing 10% FBS at a density of 150,000 cells/well until they reached 80–90% confluency, and then serum-deprived for 48 h in DMEM. A, Cells were treated with different concentrations of PE (2, 5, and 10 μM) for 18 h prior to the addition of [ 3 H]thymidine (0.50 μCi/ml) for the last 4 h. B, Cells were treated with PE for different incubation periods prior to the addition of [ 3 H]thymidine (0.50 μCi/ml). Cells were incubated with [ 3 H]thymidine for the last 4 h and thymidine incorporation was determined as described in Methods. Data are expressed as dpm of [ 3 H]thymidine incorporated in DNA per well. Values are the mean ± S.E. of six independent experiments performed in quadruplicates on different batches of cells. * Values significantly different from vehicle, p < .05. To determine whether PE at different concentrations delays the re-entry of rat-1 cells into the cell cycle, we examined the kinetics of cell cycle re-entry. Flow cytometry studies showed that incubation of the rat-1 cells with 2, 5, and 10 μM PE resulted in inhibition of the re-entry of 83, 81, or 85%, respectively, of the cell population into the S phase of the cell cycle (Table 1 ). Therefore, stimulation of α 1A -AR inhibits DNA synthesis at the G 1 /S checkpoint in rat-1 fibroblasts. Table 1 Effect of PE on cell cycle phases. Treatment G 0 /G 1 phase S phase G 2 /M phase % inhibition (S) vehicle 9269 ± 0.5 319.5 ± 13.5 238.5 ± 23.5 0 % 2 μM PE 9770 ± 9.0 54.0 ± 2.0 125 ± 21.0 83% 5 μM PE 9699 ± 29.0 59.0 ± 4.0 170 ± 11.0 81% 10 μM PE 9761.5 ± 89.5 47.5 ± 13.5 110.5 ± 39.5 85% Cells were subjected to flow cytometric DNA analysis as described in Methods. Different concentrations of PE inhibited the re-entry of the cell population into S phase of the cell cycle. Data show the number of cells in each phase of the cell cycle. Values are the mean ± S.E. of three independent experiments performed in duplicate on different batches of cells (Total events per cycle is 10,000 cells). Stimulation of α 1A -AR increases protein synthesis and promotes hypertrophy in rat-1 fibroblasts Stimulation of α 1 -ARs in vascular smooth muscle cells [ 19 ] and in adult rat ventricular myocytes [ 8 ] increases hypertrophy and protein synthesis. However, the α 1 -AR subtype mediating these effects is not known. Exposure to 5 μM PE for 18 h increased [ 3 H]leucine incorporation by 64% (Fig 2B ) and decreased basal [ 3 H]thymidine uptake (Fig 2A ), as shown earlier in Fig 1 . These effects were blocked by prazosin (PRZ, 100 nM), an α 1 -AR antagonist. Propranolol (2 μM), a β-AR antagonist (Fig 2A,B ), or yohimbine (1 μM), an α 2 -AR antagonist (data not shown), had no effect on the decrease in basal [ 3 H]thymidine or the increase in [ 3 H]leucine incorporation in rat-1 cells caused by exposure to PE. These results show that PE stimulates protein synthesis and inhibits DNA synthesis in rat-1 cells through activation of α 1A -ARs. The hypertrophy index, an indicator of cellular hypertrophy, is defined as the ratio of protein synthesis ([ 3 H]leucine incorporation) to DNA synthesis ([ 3 H]thymidine incorporation) [ 20 ]. At 18 h, this ratio was 4.4 fold as high in PE-treated cells (index = 0.22) than in control cells (index = 0.05, n = 6), indicating that PE promotes rat-1 cell hypertrophy. Figure 2 Stimulation of α 1A -AR mediates the effect of PE on DNA and protein synthesis. A, Effect of prazosin (α 1 -AR antagonist) and propranolol (β-AR antagonist) on PE-induced inhibition of DNA synthesis. Cells were treated with prazosin and/or propranolol for 30 min before the addition of 5 μM PE for 18 h. [ 3 H]thymidine incorporation was measured as described in Methods. Data are expressed as dpm of [ 3 H]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed in quadruplicates on different batches of cells. * Value significantly different from the corresponding value obtained without PE treatment, p < .05. B, Effect of prazosin and propranolol on PE-induced increase in protein synthesis. Experimental conditions were similar as described in A. [ 3 H]leucine incorporation was measured as described in Methods. Data are expressed as dpm of [ 3 H]leucine incorporated per well. Values are the mean ± S.E of three independent experiments performed in quadruplicates on different batches of cells. * Value significantly different from the corresponding value obtained without PE treatment, p < .05. Protein synthesis elicited by α 1 -AR stimulation was time-dependent and remained significant from 6 to 72 h of exposure with PE (Fig 3A ). EGF and cAMP have opposite effects on rat-1 fibroblasts proliferation [ 21 , 22 ]. Therefore, we tested the effect of EGF and forskolin (FN), an activator of adenylyl cyclase [ 12 ], on protein and DNA synthesis. EGF (30 ng/ml) slightly increased, whereas FN decreased, [ 3 H]leucine incorporation at 6, 18, 48 and 72 h (Fig 3A ). Surprisingly, PE was a better activator of protein synthesis than EGF. In contrast, EGF increased [ 3 H]thymidine incorporation by 272% (Fig 3B ), an effect blocked by FN and the cAMP analog, 8-cpt-cAMP, as well as by PE. Figure 3 Effect of PE, cAMP-elevating agents and EGF on protein and DNA synthesis . A, Cells were treated with 5 μM PE, 1 μM forskolin (FN) and 30 ng/ml EGF for different periods. [ 3 H]leucine incorporation was measured as described in Methods. Data are expressed as dpm of [ 3 H]leucine incorporated per well. Values are the mean ± S.E. of three independent experiments performed in sextuplicates. * Values significantly different from vehicle, p < .05. B, Effect of FN, 8-cpt-cAMP and PE on EGF-induced [ 3 H]thymidine incorporation. Cells were preincubated with FN (1 μM), 8-cpt-cAMP (5 μM) and PE (5 μM) for 1 h. Cells were then treated with EGF (30 ng/ml) for 18 h. Incorporation of [ 3 H]thymidine into rat-1 fibroblasts was determined as described in Methods. Data are expressed as dpm of [ 3 H]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed in duplicate in different batches of cells. * Value significantly different from vehicle, p < .05. § Value significantly different from the corresponding value obtained without treatment by FN, cpt-cAMP or PE, p < .05. PE inhibits DNA synthesis and promotes hypertrophy by a mechanism independent of cAMP Previously, we have shown that PE induces cAMP accumulation in rat-1 cells [ 12 ]. It is also known that cAMP-dependent PKA-mediated inhibition of cell division is due to blockade of growth factor-stimulated cell cycle progression from G 1 to S phase [ 23 ]. Therefore, to determine whether cAMP mediates the antiproliferative effect of PE, we examined the effect of FN (0.1–10 μM) and 8-cpt-cAMP (5–20 μM) and of FN and PE on DNA synthesis in the presence and absence of adenylyl cyclase and PKA inhibitors. FN and 8-cpt-cAMP inhibited [ 3 H]thymidine incorporation to the same extent as PE (Fig 4A ). The FN but not the PE-induced decrease in [ 3 H]thymidine incorporation was blunted by the selective inhibitor of adenylyl cyclase SQ 22536 (Fig 4B ) and by the P-site inhibitors of adenylyl cyclase 2',5'dideoxyadenosine, and 2'-deoxyadenosine 3'-monophosphate (5 μM each-data not shown). The selectivity of cAMP modulators, FN, 8-cpt-cAMP and SQ 22536 has been tested previously in rat-1 fibroblasts [ 12 ]. To further determine the contribution of cAMP pathway, rat-1 cells were transfected with πLXX-PKI [ 1 - 31 ], a vector that expresses the PKA inhibitor, PKI [ 23 , 24 ]. Transfection with PKI reversed the inhibitory effect of FN, but not that of PE, on [ 3 H]thymidine incorporation in rat-1 cells (Fig 4C ). These observations indicate that cAMP and PKA do not mediate the PE-induced inhibition of DNA synthesis. Figure 4 Effect of PE and cAMP-elevating agents, FN and 8-cpt-cAMP, on DNA synthesis . Cells were serum-deprived for 2 days. A, Cells were treated with PE (5 μM), FN (0.1, 1, and 10 μM), and 8-cpt-cAMP (5, 10, and 20 μM) for 18 h. Incorporation of [ 3 H]thymidine into rat-1 fibroblasts was determined as described in Methods. Data are expressed as dpm of [ 3 H]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed in sextuplicates in different batches of cells. * Value significantly different from vehicle, p < .05. B, Effect of inhibitors of adenylyl cyclase inhibition on PE and cAMP-elevating agents. Cells were pre-incubated with SQ22536 (25 μM), an specific adenylyl cyclase inhibitor for 1 h. Cells were then treated with 5 μM PE and/or 1 μM FN for 18 h. [ 3 H]thymidine incorporation was determined as described in Methods. Values are the mean ± S.E. of three independent experiments performed in quadruplicates on different batches of cells. Data are expressed as dpm of [ 3 H]thymidine incorporated per well. * Value significantly different from the corresponding vehicle, p < .05. § value significantly different from that obtained in the presence of agonist alone, p < .05. C, Effect of transfection of πLXX-PKI [1-31] on PE-induced inhibition of DNA synthesis. Preconfluent cells were transiently transfected with πLXX-PKI [1-31], a plasmid encoding for the protein kinase inhibitor, (PKI), using Lipofectamine Plus transfection reagent according to the manufacturer's protocol (Life Technologies). Cells were cultured in serum-free DMEM for 24 h and then treated with 5 μM PE and/or 1 μM FN. Cells were assayed for [ 3 H]thymidine incorporation 48 h post transfection as described in Methods. Data are expressed as dpm of [ 3 H]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed on different batches of cells. * Value significantly different from the corresponding vehicle, p < .05. § value significantly different from that obtained in the presence of agonist alone, p < .05. The antiproliferative effect of PE, FN and 8-cpt-cAMP could be the result of cell loss due to a cytotoxicity. However, the trypan blue exclusion test and LDH assay showed that more than 90% of cells were viable (Table 2 ) and less than 1% of the cells were found in the supernatant, indicating very little cell loss. Table 2 Effect of PE and cAMP elevating agents on viability of rat-1 fibroblasts using the hemocytometer trypan blue method. Treatment Total number of cells Blue cells % of viability Vehicle 78 8 90% 5 μM PE 142 10 92% 10 μM PE 87 12 86% 10 μM FN 78 8 90% 20 μM 8-cpt-cAMP 84 4 96% 20 μM 8-Br-cAMP 66 2 97% Cells were incubated for 18 h with PE (5 and 10 μM), forskolin (FN), 8-cpt-cAMP and 8-Br-cAMP. The percentage of viable cells was calculated. Values are representative of five different experiments. Morphological change elicited by α 1A -AR stimulation and by cAMP in rat-1 cells Although both PE and FN inhibited DNA synthesis in rat-1 cells, they produced different effects on cell morphology. PE increased, whereas FN decreased, the apparent size of rat-1 cells as shown in Fig 5 . Prazosin (100 nM), an α 1 -AR antagonist, reversed the shape change produced by PE. To determine if PE-induced cell cycle arrest and hypertrophy of rat-1 fibroblasts is also independent of cAMP, we preincubated the cells with SQ22536 (25 μM) and then treated them with PE (5 μM) or FN (1 μM) for different time periods. SQ22536 reversed the morphological changes produced by FN and restored the normal fibroblast shape, whereas it enhanced the effect of PE, i.e. the cells became more hypertrophic. Figure 5 Effect of prazosin and SQ22536 on PE-and FN-induced morphological change in rat-1 cells . Preconfluent, serum-deprived cells were either stimulated with 5 μM PE and/or 1 μM FN for different periods of time (Fig 5B) and/or pretreated for 30 min with 100 nM prazosin (Fig 5A) or 25 μM SQ22536 (Fig 5C), and then further incubated with 5 μM PE and/or 1 μM FN for 24 h. Morphological studies were performed as described in Methods. The same morphological patterns were obtained in three different experiments. p27 kip1 mediates PE-induced inhibition of DNA synthesis Cyclin-dependent kinase (cdk) inhibitors p27 kip1 and p21 cip1/waf1 play an important role in cell-cycle regulation [ 25 , 26 ]. High levels of p27 kip1 and p21 cip1/waf1 in quiescent (G 0 ) cells decline upon induction of mitogenesis [ 27 ]. This decrease in Cdk inhibitors appears to be critical in enabling cell cycle entry. Western blot analysis showed that α 1A -AR stimulation increased the expression levels of p27 kip1 (642% of control at 48 h) and p21 cip1/waf1 (935% of control at 48 h) (Fig. 6A ). The protein levels remained elevated for 48 h, with levels of p27 kip1 reaching 389% of control at 18 h of incubation and remaining elevated for up to 48 h. This increase was concomitant with the inhibitory effect of PE on DNA synthesis, which was also maximal at 18 h of incubation. PE increased the expression of p27 kip1 (552% of control at 24 h) more efficiently than p21 cip1/waf1 (87% of control at 24 h) suggesting a greater contribution of p27 kip1 to PE-induced cell cycle arrest and a delayed induction of p21 cip1/waf1 . Retinoblastoma protein (pRb) hyperphosphorylation and subsequent release of E2F is required for cell cycle progression [ 28 ]. However, α-AR stimulation of neonatal myocytes has been shown to induce pRb hyperphosphorylation without induction of cell proliferation [ 29 ]. Our results demonstrate that PE promotes an early increase in pRb levels (607% of control at 6 h) in rat-1 cells (Fig 6C ), associated with a decrease in DNA synthesis (Fig 1B ). The nuclear envelope proteins lamin A and C, used as control, were not altered by PE treatment (Fig 6D ). Figure 6 Effect of PE on the expression of cell cycle regulators, p27 kip1 , p21 ip1/waf1 and pRB . Preconfluent, serum-deprived cells were incubated with PE (5 μM) or vehicle for up to 48 h. Expression of cell cycle regulators was determined by Western blot analysis of whole cell lysates as described in Methods. The panels are representative Western blots showing the effect of PE on the expression of p27 kip1 (A), p21 cip1/waf1 (B), pRb (C), and lamin A/C (D) protein levels respectively. Percentages representing the quantitation of protein bands by densitometric analysis of blots obtained from three different experiments are indicated on top of each immunoblot. In VSMC, p27 kip1 level is a critical determinant of the cell response, hyperplasia vs. hypertrophy, to angiotensin II and other growth factors [ 20 ]. We postulated that the response of rat-1 cells to PE was mediated by p27 kip1 . PE upregulates the expression of p27 kip1 at 18 h of treatment in rat-1 cells at all concentrations (Fig 7A ). We therefore explored further the role of p27 kip1 in modulating the antiproliferative response of rat-1 cells to PE by transfecting these cells with p27 kip1 antisense and sense ODN. p27 kip1 antisense (102% of control), but not sense ODN (347% of vehicle vs. 338% for PE alone), diminished p27 kip1 level (Fig 7B ) and blocked the antiproliferative effect of PE in rat-1 cells (Fig 7C ). These observations suggest that PE-induced inhibition of DNA synthesis is mediated by p27 kip1 . Figure 7 Effect of PE on p27 kip1 level and effect of p27 kip1 antisense oligonucleotides on PE-induced inhibition of DNA synthesis . A, Preconfluent rat-1 cells were stimulated with 2, 5, and 10 μM PE for 18 h. Total cell lysates were subjected to Western blot analysis as described in Methods. Percentages representing the quantitation of protein bands by densitometric analysis of blots obtained from three different experiments are indicated on top of each immunoblot. B, Representative Western blot showing the effect of p27 kip1 antisense and sense oligonucleotides treatment on PE-induced increase in p27 kip1 protein level. Cells were transiently transfected with p27 kip antisense or sense oligonucleotide for 48 h using Oligofectamine™ Reagent according to the manufacturer's instruction (Invitrogen). C, Transfected cells as in B were serum-starved for 24 h and then treated with 5 μM PE. Thymidine uptake was measured as described in Methods 48 h post-transfection. Data are expressed as the percent decrease in [ 3 H]thymidine uptake above the basal value obtained in unstimulated cells. Values are the mean ± S.E of three independent experiments performed in different batches of cells. * Value significantly different from the corresponding vehicle, p < .05. PE increases the expression of cyclin D1, proliferating cell nuclear antigen (PCNA), Cdk2 and Cdk4 in rat-1 cells We next investigated the effect of PE on several cell cycle proteins important for G 1 /S phase progression. Fig. 8 shows that treatment of serum-deprived rat-1 cells with PE increased the levels of cyclin D1 (548% of control at 24 h), PCNA (158% of control at 24 h), and Cdk2 (236% of control at 24 h) in a time-dependent manner. The levels of nuclear envelope proteins lamin A and C, were not altered. These results are unexpected since PE promotes cell cycle arrest at G 1 /S checkpoint (Table 1 ). However, the up-regulation of p27 kip1 seems to be sufficient to block cell cycle entry into S phase and DNA synthesis. Surprisingly, we also observed an increase in the protein levels of Cdk4 (280% of control at 24 h), which normally do not change even during mitogenic stimulation. Figure 8 Effect of PE on the expression of cyclin D1, PCNA Cdk 2, and Cdk 4 . Preconfluent, serum-deprived cells were stimulated with PE (5 μM) for 6, 12, 24 and 48 h. Cell-cycle regulators expression was determined by Western blot analysis of whole cell lysates as described in Methods. The bar graph represents quantitation of protein bands by densitometric analysis of blots obtained from three different experiments. * Value significantly different from the corresponding vehicle, p < .05. PE increases expression of specific smooth muscle differentiation markers as well as MyoD Cell cycle arrest is closely coupled to muscle cell differentiation and is required for activation of muscle cell specific gene expression [ 16 ]. Since stimulation of α 1A ARs promoted a change in morphology associated with hypertrophy, we examined the effect of PE on expression of the specific smooth muscle cell markers, α-smooth muscle actin (SMα actin), caldesmon and myosin heavy chain (SM-MHC) [ 30 ]. The smooth muscle markers were not expressed in serum-deprived rat-1 cells (Fig 9A,C ). Stimulation of α 1A ARs with PE (5 μM) promoted a time-dependent increase in the levels of SMα actin (695% of control at 24 h) (Fig 9A ), caldesmon (284% of control at 24 h) (Fig 9B ) and SM-MHC (445% of control at 24 h) (Fig 9C ) as shown by Western blot analysis We also investigated the expression of MyoD, a helix-loop-helix protein that plays an important role in the regulation of muscle development [ 31 ]. Western blot analysis showed that PE also increased the expression of MyoD in a time-dependent manner (457% of control at 24 h), further supporting the evidence that stimulation of α 1A AR induces differentiation of rat-1 fibroblasts into muscle-like cells, which is closely coordinated with cell cycle arrest (Fig 9D ). The expression of vimentin, an intermediate filament protein used as a control, was not altered by treatment with PE (Fig 9E ). The α 1A AR stimulation of smooth muscle differentiation markers was independent of cAMP because FN did not increase the expression of SMα actin, caldesmon or SM-MHC (Fig 10A,C ). MyoD expression was not altered by FN (Fig 10D ). The level of vimentin expression was not altered by PE or FN (Bar Graph). Together, these data demonstrate that stimulation of α 1A ARs increases expression of smooth muscle markers and MyoD in rat-1 cells, independently of cAMP. Figure 9 Time course of the effect of PE on the expression of the smooth muscle differentiation markers and MyoD . Preconfluent, serum-deprived fibroblasts were stimulated with PE (5 μM) for up to 48 h. The protein expression of smooth muscle differentiation markers and MyoD was determined by Western blot analysis of whole cell lysates as described in Methods. A, smooth muscle α actin (SMα actin). B, caldesmon. C, smooth muscle myosin heavy chain (SM-MHC). D, MyoD, E, vimentin. VSMC lysate is shown as a positive control. Percentages representing the quantitation of protein bands by densitometric analysis of blots obtained from three different experiments are indicated on top of each immunoblot. Figure 10 Effect of PE and FN on the expression of smooth muscle differentiation markers and MyoD. Preconfluent, serum-deprived fibroblasts were stimulated with either PE (5 μM) or FN (1 μM) for up to 48 h. Expression of markers was determined by Western blot analysis of whole cell lysates as described in Methods. A, smooth muscle α actin (SMα actin). B, caldesmon. C, smooth muscle myosin heavy chain (SM-MHC). D, MyoD. VSMC lysate is shown as a positive control in panel A, B, C and D. The bar graph represents quantitation of protein bands by densitometric analysis of blots obtained from three different experiments. * Value significantly higher than the corresponding vehicle, p < .05. Discussion This study is the first demonstration of the ability of an α 1A -AR subtype to promote differentiation of a fibroblast into a SMC/myocyte-like cell. Further, it demonstrates that α 1A -AR-induced cell cycle arrest and hypertrophy as well as differentiation is mediated through a mechanism dependent upon the increased expression of the critical cell cycle protein p27 kip1 and selective smooth muscle markers. Stimulation of α 1A -ARs promotes hypertrophy of cardiac myocytes [ 6 ]. Norepinephrine increases SMC hypertrophy, but not cell proliferation, through α 1A -AR stimulation in uninjured aorta [ 8 ]. In HepG2 and M12 cells transfected with α 1A -AR, PE stimulates cell scattering and inhibition of proliferation [ 15 ]. Similar observations have been made in CHO cells expressing α 1A -ARs [ 17 , 18 ]. In the present study, PE inhibited cell proliferation, promoted hypertrophy and differentiation through stimulation of α 1A -ARs in rat-1 cells expressing this subtype. Analysis of the effect of α 1A -AR stimulation on cell cycle progression revealed that it inhibits the re-entry of cells from G 0 /G 1 into the S phase of the cell cycle. Next we attempted to define the mechanism mediating α 1A -AR-induced cell cycle arrest, hypertrophy and differentiation of rat-1 cells. Previous studies have shown that in rat 1-cells expressing α 1A -ARs, PE promotes increase in cAMP accumulation and PKA activation [ 12 ]. Since cAMP has been reported to inhibit growth in various cell types, including cells of fibroblastic origin [ 32 ], it seemed possible that cAMP mediates the inhibitory effect of PE on rat-1 cells proliferation. Stable cAMP analogs or adenylyl cyclase activation promoted cell cycle arrest similar to α 1A -AR stimulation. However, activation of the cAMP/PKA pathway did not promote hypertrophy or differentiation of rat-1 cells. Moreover, inhibition of cAMP/PKA did not reverse the α 1A -AR-induced inhibition of DNA synthesis. Therefore, α 1A -AR-induced cell cycle arrest, hypertrophy and differentiation, is independent of the cAMP/PKA pathway in rat-1 cells. Recently, the cAMP pathway has been implicated in α 1A -AR-induced cell cycle arrest in CHO cells expressing α 1A -ARs [ 18 ]. The difference between these cell lines may be due to the inhibitory effect of PE on ERK in rat-1 cells [ 33 ] vs. PE-induced ERK activation in CHO cells [ 18 ]. In addition, the potential effect of α 1A -AR stimulation on CHO cells hypertrophy and differentiation is not known. During our investigation of the possible mechanism by which PE induces cell cycle arrest, we observed that although both PE and FN inhibited DNA synthesis, these two agents produced remarkably different morphological changes in rat-1 cells. FN promoted a change in rat-1 cells to a small, shrinked, round shaped morphology whereas PE shifted the cells to a hypertrophied, enlarged and spindle shaped smooth muscle-like phenotype. This α 1A -AR-induced morphological change was again independent of cAMP. Differences between PE and cAMP on different cell functions are reported in Table 3 . EGF increases DNA synthesis in rat-1 fibroblasts [ 22 ] and slightly increased protein synthesis (our data). However, EGF was a far less potent hypertrophic agent than PE and did not change the morphology of rat-1 cells. Table 3 Comparison of the effects of PE and FN on different cell parameters. Parameter PE FN cAMP production ↑ ↑ Protein synthesis ↑ ↓ DNA synthesis ↓ ↓ Morphology enlarged; spindle-shaped shrunk; rounded Differentiation markers ↑ ↓ Cdk inhibitors ↑ ↑ The arrows indicate a significant increase or decrease elicited by PE or FN in the corresponding cell parameter studied. These agents' stimulated cAMP production, decreased basal DNA synthesis, and up-regulated cyclin-dependent kinase inhibitors. PE and cAMP have opposite effects on protein synthesis, cell morphology and smooth muscle differentiation markers. The different effects of α 1A -AR stimulation and cAMP on rat-1 cell hypertrophy and morphology led us to explore the effects of PE and FN on different cell parameters associated with morphological changes, i.e. cell cycle regulators and smooth muscle cell differentiation markers. Recently, it has been reported that the Cdk inhibitor p27 kip1 mediates angiotensin II-induced cell cycle arrest and hypertrophy in cultured renal tubular cells [ 34 ], and vascular smooth muscle cells [ 20 ] and induces intestinal epithelial cell differentiation [ 35 ]. In the present study, stimulation of α 1A -ARs increased the expression of p27 kip1 to a much greater extent than p21 cip1/waf1 , suggesting an important role for the former Cdk inhibitor in the action of α 1A -AR stimulation. Interestingly, the time of upregulation of p27 kip1 by PE correlated closely with its effect on inhibition of DNA synthesis, which was maximal at 18 h. Depletion of p27 kip1 prevented both PE-induced upregulation of p27 kip1 and reversed PE-induced decrease in DNA synthesis. Therefore, p27 kip1 plays a central role as a mediator of α 1A -AR-induced inhibition of DNA synthesis and probably hypertrophy and differentiation of rat-1 fibroblasts. An important finding in the present study was that PE produced a change in the morphology of rat-1 cells characterized by an increase in cell size. Supporting this phenotypic and hypertrophic change was our demonstration that PE increased global protein synthesis and the expression of markers specific to smooth muscle cells such as smooth muscle actin, caldesmon, and myosin heavy chain. The protein levels of these smooth muscle cell markers were found to remain elevated for up to 48 h, consistent with the PE-induced morphological change that persisted for the same time period. Although stimulation of α 1A -ARs shifted rat-1 fibroblasts to SMC/myocyte-like phenotype, it also increased the expression of the helix loop helix protein MyoD, a skeletal muscle-specific regulatory transcription factor [ 31 ]. Surprisingly, PE also caused an increase in pRb expression to a level similar to p27 kip1 . MyoD has been shown to interact with pRb and to promote muscle gene activation and cell cycle arrest [ 28 , 36 ]. Indeed, pRb has been found to contain a differentiation-promoting activity that is distinct from its cell-cycle progression functions [ 37 ]. More recent evidence has indicated an essential role for pRb in promoting functional synergism between MyoD and MEF2 proteins [ 38 ]. Although PE increased the expression of MyoD in our study, the rat-1 cells had a smooth muscle/myocyte-like phenotype. It has been reported that vascular smooth muscle cells can spontaneously adopt the skeletal muscle phenotype [ 31 ]. Therefore, it is possible that PE increases expression of MyoD in rat-1 cells during differentiation into myocyte-like cells. However, expression of MyoD is not sufficient for the coordinated program of skeletal myogenesis in smooth muscle cells [ 31 ]. PE-induced hypertrophy and differentiation of rat-1 cells was also associated with increased levels of cell cycle proteins pRb, cyclin D1, PCNA and Cdk2 that are important for G 1 /S phase progression [ 39 ]. These data indicate that stimulation of α 1A -ARs promotes the transcriptional/translational activation of the machinery required for G 1 /S cell cycle progression. However, a simultaneous increase in Cdk inhibitors such as p27 kip1 prevented DNA synthesis. Elevation of p27 kip1 protein level alone is sufficient for induction of cell cycle arrest, independent of cyclins or Cdk level [ 40 ]. Surprisingly, PE also increased the protein level of Cdk4, which is normally not affected by mitogenic stimuli. Although the Cdk inhibitors bind to cyclin/Cdk complexes and reduce their activity, their interaction is probably much more complex [ 25 , 26 ]. Cdk inhibitors may paradoxically activate these kinases, particularly cyclin D/Cdk4, 6 complexes [ 26 ]. The mechanism by which stimulation of the α 1A -AR promotes the up-regulation of Cdk inhibitors (p27 kip1 , p21 cip1/waf1 ), smooth muscle cell markers, MyoD, and G 1 /S transition cell cycle proteins (pRb, cyclin D1, PCNA, Cdk2/4) leading to inhibition of proliferation and stimulation of hypertrophy and differentiation is not known. The cAMP/PKA pathway was excluded by our results as well as the ERK pathway [ 33 ]. The phosphatidylinositol 3-kinase and Akt/PKB pathway, a pro-survival/mitogenic and hypertrophic pathway is also unlikely to be involved, because PE does not stimulate this pathway in rat-1 cells [ 42 ]. Recently, a study on the genetic profiling of rat-1 fibroblasts expressing different subtypes of α-AR has shown that in cells expressing the α 1A -AR, epinephrine (one hour stimulation) increased the gene expression of IL-6, gp-130 (an IL-6 high affinity receptor and signal transducer) and STAT-3 (an IL-6 activated transcription factor) [ 42 ]. Moreover, in cells expressing α 1A -adrenergic receptor, epinephrine also increased IL-6 secretion and STAT-3 Ser 727 phosphorylation [ 42 ]. Therefore, it is possible that IL-6, gp130, and/or STAT-3 contributes to the upregulation of one or more of the cell cycle associated proteins and smooth muscle cell markers responsible for the cells arrest, hypertrophy and/or differentiation caused by α 1A -AR activation in rat-1 cells. With regard to functional significance in vivo , our model uses a transformed embryonic fibroblast cell line that expresses high levels of α 1A -AR [ 12 ]. Therefore, the relevance of these results to fibroblasts in tissues remains to be determined. These features in our model may underlie the ability of PE to cause expression of SMC markers and MyoD through α 1A -AR. Conclusions This study demonstrates that stimulation of α 1A -ARs in rat-1 cells promotes cell cycle arrest by increasing levels of Cdk inhibitors and promotes hypertrophy and differentiation into a phenotype having the characteristics of smooth muscle cells by a mechanism independent of cAMP or EGF. Moreover, cell cycle progression was blocked at G 1 /S transition without causing apoptosis, and this cycle arrest was critical for rat-1 cell hypertrophy and differentiation. Reducing p27 kip1 levels reversed α 1A -AR-promoted inhibition of DNA synthesis. Furthermore, it shows that cell cycle arrest and differentiation are closely coordinated processes but temporally separable. Further studies are underway in our laboratory to characterize the signaling pathway(s) involved in α 1A -AR-induced differentiation of fibroblasts to smooth muscle cells. Methods Materials [Methyl- 3 H]thymidine (20 Ci/mmol) was purchased from NEN Life Science Products, Inc. (Boston, MA). L-[4,5- 3 H] leucine from Amercham Pharmacia Biotech. (Piscataway, NJ.). L-phenylephrine hydrochloride, penicillin, streptomycin, prazosin, propranolol, epidermal growth factor (EGF), and propidium iodide were obtained from Sigma (St. Louis, MO). Forskolin (FN), 8-cpt-cAMP and SQ 22536 were purchased from Calbiochem (La Jolla, CA). 2'-5'dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphates were purchased from Biomol (Playmouth Meeting, PA). G418 sulfate from Invitrogen (Carlsbad, CA). Hanks' balanced salt solution and fetal bovine serum were from Mediatech, Inc. (Herndon, VA). Dulbecco's Modified Eagle's Medium (DMEM) and trypsin/EDTA were obtained from Life Technologies Inc. (Grand Island, N.Y). Rat-1 cells and culture conditions Rat-1 cells transfected with bovine α 1A -AR kindly provided to us by Drs. L. Allen, R. J. Lefkowitz, and M. G. Caron (Duke University), were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum, 400 μg/ml G418 sulfate, 50 μg/ml streptomycin and 50 units/ml penicillin at 37°C in an humid atmosphere (5% CO2, 95% air). Cells were serially passaged upon reaching confluence, and all experiments were performed on subculture passages 5–15. Prior to all experiments, preconfluent cell cultures were serum-starved for 48 h. Measurement of DNA synthesis Cells at a density of 150,000 cells/well were seeded in DMEM containing 10% FBS on 24-well plates until they reached 80–90% confluency and were serum-deprived for 48 h prior to the addition of agonists. Rat-1 cells, even after serum-deprivation for 2 days, exhibit a measurable level of [ 3 H]thymidine incorporation. Inhibition of [ 3 H]thymidine incorporation was used as a quantitative measure of reduction in DNA synthesis. Cells were serum-starved in the presence or absence of agonists for the indicated time period, and [ 3 H]thymidine (0.5 μCi/ml) was added for the last 4 h of the incubation. This time period was chosen because it gave the maximum incorporation of [ 3 H]thymidine into DNA after addition of agonists. The medium was then removed, and the cells were washed twice with phosphate-buffered saline and three times with 10% ice-cold trichloroacetic acid. Cells were kept in contact with trichloroacetic acid for 10 min during each wash. The precipitated material was dissolved in 1 M NaOH containing 0.1% SDS, and radioactivity was determined by liquid scintillation spectrometry. Measurement of protein synthesis Rat-1 cells were seeded at a density of 150,000 cells/well in 24-well plates and just prior to confluency were serum-deprived for 48 h. Cells were rinsed with DMEM and 1 μCi/ml [ 3 H]leucine was added for the last 4 h of each incubation with or without the indicated concentrations of the agonists. Cells were fixed and washed three times with ice-cold trichloroacetic acid (10%). The precipitated material was solubilized with 0.1 M NaOH and the incorporated radioactivity was counted by liquid spectrometry. Flow cytometry Flow cytometry studies were performed to determine the effect of different concentrations of PE on cell cycle phases. Cells were subjected to flow cytometric DNA analysis as described [ 43 ] with some modifications. Briefly, rat-1 cells plated on 100 mm dishes were grown in DMEM containing 10% FBS until they reached 80–90% confluency. Preconfluent cell cultures were serum-starved for 48 h to stop the mitogenic effect of growth factors. The medium was aspirated and cells were incubated for 18 h with different concentrations of PE. Cells were trypsinized in 1 ml trypsin/EDTA for at least 5 min, and then the reaction was stopped by the addition of 1 ml of serum-containing DMEM. The samples were centrifuged at 1000 rpm for 5 min, washed three times in ice-cold PBS containing 1% bovine serum albumin by centrifugation at 1000 rpm for 5 min each, resuspended in 0.5 ml of the same solution, and then fixed with 1 ml of 70% ethanol (-20°C) added dropwise. The fixed cells were stored at 4°C until analyzed. Cells were then washed three times by centrifuging at 1000 rpm for 10 min and resuspended in 3 ml of the BSA buffer. The pellet was then finally resuspended in 1 ml of the BSA buffer to which 100 μg/ml of RNAse A was added to remove interfering RNA, and 5 μg/ml propidium iodide was added to stain DNA. Cells were incubated at 37°C for 10–15 min in the dark to facilitate staining. The cells were analyzed for DNA content using an Epics Profile Analyzer (Coulter Electronics) with an Argon laser emitting at 448 nm. Percentages of cells in various stages of the cell cycles were determined using a multi-cycle program (P. Rabinovitch, Phoenix Flow Systems). At least 10,000 cells per cycle were counted for each treatment. Cell viability Preconfluent cultures were serum-starved for 48 h. Cells were incubated with different concentrations of PE, FN and 8-cpt-cAMP for 18 h, trypsinized with 1 ml trypsin/EDTA for 5 min and centrifuged for 10 min at room temperature. The pellet was resuspended in 1 ml plain DMEM. 10 μL of the suspension was mixed with 10 μL of 0.4% trypan blue in a 0.5 ml microtube. The total number of cells and the number of blue cells and the percentage of the viable cells in 10 μL of the trypan blue mixture was calculated as follows: % viable cells = [1-(blue cells/total cells)] × 100. Morphological study Rat-1 cells resuspended in culture medium were seeded on six-well plates (Corning, N.Y.). Preconfluent cells were serum-deprived for 2 days, pre-incubated with inhibitor (s) for 30 min, and then stimulated with 5 μM PE and /or 1 μM FN for the indicated time. Cells were rinsed twice in PBS to remove non-adherent cells and then fixed for 10 min in [1:1] methanol-acetone mixture at room temperature. Cells were washed once in distilled water and stained in hematoxylin solution (Sigma, St. Louis, MO) for 15 min at room temperature. Cells were again washed three times in water, air dried and were finally observed under a phase contrast microscope. Images were captured and saved as TIFF files. For each condition, data were collected by random observation. Hypertrophic phenotype was defined as both enlarged, elongated and spindle-shaped whereas rat-1 fibroblasts are round and/or polygonal-shaped cells. Transfection procedures To determine whether protein kinase A (PKA) mediates the antiproliferative effect of PE, rat-1 cells were transiently transfected with πLXX-PKI [ 1 - 31 , 24 ], a plasmid encoding for the PKA inhibitor, PKI (A generous gift from Dr. J. Avruch, Massachusetts General Hospital, Boston, MA), using Lipofectamine Plus (Life Technologies, Inc., Grand Island, N.Y.) according to the manufacturer's instructions. For p27 kip1 experiments, phosphorothionate oligonucleotides (ODN) (Life Technologies, Grand Island, N.Y.) were used. The antisense ODN sequence used in the experiments was 5'-CACTCTCACGTTTGACAT-3' (nuc 1–18 of rat p27 kip1); the sense ODN sequence was 5'-ATGTCAAACGTGAGAGTG. ODN transfection was performed with oligofectamine in Opti-MEM according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Cells were left in the transfection mixture for 48 h and then incubated for 24 h with or without agonists and harvested for Western blot, or labeled with [ 3 H]thymidine to determine DNA synthesis. Preparation of cell lysates and western blot analysis Cells were rinsed twice with PBS and lysed in ice-cold lysis buffer (1% Igepal CA-630, 25 mM HEPES pH 7.5, 50 mM NaCl, 50 mM NaF, 5 mM EDTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 5 mg/ml pepstatin A, 100 mM PMSF, 100 mM sodium orthovanadate, and 10 μM okadaic acid). Lysates were centrifuged at 4°C for 15 min in a microfuge at maximum speed, and the supernatant was collected for Western blot analysis. Equal amounts of protein were separated on denaturing SDS/polyacrylamide gel and transferred on nitrocellulose blots (Hybond-ECL; Amersham Life Sciences Inc., Arlington Heights, IL) by electrophoresis. Blots were blocked for 1 h in 5% nonfat dry milk in TBST, washed three times 5 min each in TBST and then incubated with the indicated specific primary antibody overnight at 4°C: p27 kip1 , p21 waf1/cip1 , pRb (Biosource International); caldesmon (smooth muscle), myosin (smooth muscle), α-smooth muscle actin, β-actin (Sigma Biosciences); MyoD, cyclin D1, Cdk 2, Cdk 4, PCNA, lamin A/C or vimentin (Santa Cruz Biotechnology). Following incubation, the membranes were washed three times 10 min each in TBST and incubated with a secondary antibody coupled to peroxidase for 1 h at room temperature. Then the membranes were washed three times 10 min each in TBST. Specific proteins were detected by enhanced chemiluminescence (ECL; Amersham Life Sciences Inc.) according to the manufacturer's instructions and analyzed with an Alpha Innotech Fluorochem imaging system (Packard Canberra). A lysate from cultured aortic vascular smooth muscle cell (VSMC) was used as a control for the expression of smooth muscle markers. Statistical analysis The basal values of incorporation of [ 3 H]thymidine and/or leucine were variable in different batches of cells. However, the effect of various agents on the incorporation of [ 3 H]thymidine and [ 3 H]leucine in rat-1 cells was consistent within each batch of cells. The results are expressed as mean ± SEM. The data were analyzed by one-way analysis of variance; the Newman-Keuls multiple range test was applied to determine the differences among multiple groups, the unpaired Student's t-test was applied to determine the difference between two groups. The null hypothesis was rejected at p < 0.05. The protein level were estimated by densitometric analysis of the Western blots and performed on the indicated number of blots using NIH Image software, and expressed as a percentage of the control, arbitrarily chosen as 100%. Abbreviations AR, adrenergic receptor; Cdk, cyclin-dependent kinase; FN, forskolin; MHC, myosin heavy chain; PCNA, proliferating cell nuclear antigen; PE, phenylephrine; pRb, retinoblastoma protein; SMC, smooth muscle cell Authors contributions AES performed thymidine/leucine incorporation, morphological studies, cell viability, flow cytometry and Western blot analysis. JHP carried out the antisense design, transfection experiments, statistical analysis and some thymidine/leucine incorporation and Western blot analysis. KUM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548263.xml
535934	Hysterectomy at a Canadian tertiary care facility: results of a one year retrospective review	Background The purpose of this study was to investigate the indications for and approach to hysterectomy at Kingston General Hospital (KGH), a teaching hospital affiliated with Queen's University at Kingston, Ontario. In particular, in light of current literature and government standards suggesting the superiority of vaginal versus abdominal approaches and a high number of concurrent oophorectomies, the aim was to examine the circumstances in which concurrent oophorectomies were performed and to compare abdominal and vaginal hysterectomy outcomes. Methods A retrospective chart audit of 372 consecutive hysterectomies performed in 2001 was completed. Data regarding patient characteristics, process of care and outcomes were collected. Data were analyzed using descriptive statistics, t-tests and linear and logistic regression. Results Average age was 48.5 years, mean body mass index (BMI) was 28.6, the mean length of stay (LOS) was 5.2 days using an abdominal approach and 3.0 days using a vaginal approach without laparoscopy. 14% of hysterectomies were performed vaginally, 5.9% were laparoscopically assisted vaginal hysterectomies and the rest were abdominal hysterectomies. The most common indication was dysfunctional or abnormal uterine bleeding (37%). The average age of those that had an oophorectomy (removal of both ovaries) was 50.8 years versus 44.3 years for those that did not (p < .05). Factors associated with LOS included surgical approach, age and the number of concurrent procedures. Conclusions A significant reduction in LOS was found using the vaginal approach. Both the patient and the health care system may benefit from the tendency towards an increased use of vaginal hysterectomies. The audit process demonstrated the usefulness of an on-going review mechanism to examine trends associated with common surgical procedures.	Background In Canada in 2001, 446 hysterectomies were performed per 100 000 women [ 1 ]. The rate however varies considerably as a consequence of factors such as acceptability of medical management in areas where there is limited availability of gynaecologists [ 2 ] and a lack of dissemination and implementation of guidelines to direct treatment decisions [ 3 ]. In response to the consistent demand for this procedure, recent reports have identified hysterectomy as a key health care indicator used to measure and compare hospital performance. In particular, the Ontario Hospital Association has identified the ratio of vaginal (VH) to abdominal hysterectomy (AH) as a measure of hospital performance [ 4 ], with a more favorable grade awarded to those hospitals with a higher proportion of VHs. In addition, length of stay (LOS) and complication rates associated with hysterectomy are also used to grade hospital performance [ 4 ]. Considerable attention has also been directed towards the high rate of concurrent oophorectomy (removal of both ovaries) with this procedure. This rate is of particular concern in premenopausal women because of the early menopause that ensues. The purpose of this study was to compare abdominal and vaginal approaches to hysterectomy, investigate the rate of concurrent oophorectomy, and identify factors associated with length of surgery, LOS and approach, by auditing all hysterectomies performed over a one-year period at a university teaching hospital. Methods The study involved all patients who underwent a hysterectomy in 2001 at Kingston General Hospital (KGH), a teaching hospital affiliated with Queen's University at Kingston, Ontario. The Queen's University Health Sciences and Affiliated Teaching Hospitals Research Ethics Board approved the study (OBGY-117-03). There were no exclusion criteria. Patients were identified by medical record tracking using ICD-9 codes and charts were reviewed to collect patient characteristics, length of stay, length of surgery, indication for surgery and approach. Readmissions, complications, infections and repeat laparotomies were also assessed. Menopause was defined as one year since the last menstrual period. Up to three indications for surgery were obtained from the chart, including those identified in clinic letters, admission sheets and operative reports. All indications were collected regardless of whether or not the post-operative diagnosis coincided with the preoperative diagnosis. VH included laparoscopically assisted vaginal hysterectomy (LAVH) and AH included VH converted to AH unless otherwise noted. Readmission was defined as a visit to the emergency room or an admission to the same hospital with a diagnosis that was related (readmission to another facility was unlikely as KGH is the only tertiary care facility in the region). Post-operative infections were defined as those that occurred within 30 days of surgery. A complication of excessive bleeding was defined as an intra-operative hemorrhage requiring transfusion or laparotomy, post-operative hematoma/seroma formation, or a significant post-operative vaginal bleed that required medical attention. All complications that occurred during the surgery or within 30 days of surgery were recorded, other than problems associated with removal of catheter, urinary retention, hypertension, hypotension, pain control, nausea and vomiting or headache. Any repeat laparotomy or unplanned laparotomy (other than for conversion of VH to AH) that occurred during the surgery or within 30 days of discharge was also noted. Follow up information was tracked using hospital chart and clinic note information from the six-week post-operative visit. All data were analyzed using SPSS statistical software (Version 11.0.1, SPSS Incorporated, Chicago, Illinois, 2002). Between-group comparisons utilized two-sample t-tests and one-way analysis of variance (continuous data) and Chi-square analyses (categorical data). Predictors of LOS and length of surgery were identified using linear regression, while predictors of surgical approach were identified using logistic regression. Variables were offered into the models on the basis of the strength of the bivariate associations with the outcomes (p < 0.20). Results Three hundred and seventy two women underwent a hysterectomy in 2001. The characteristics of these patients can be found in Table 1 . Sixty-nine percent were premenopausal at the time of the surgery. Table 1 Patient Characteristics N Minimum Maximum Mean Std. Deviation Age (years) 372 27 87 48.5 11.5 Body Mass Index 357 16 79 28.6 7.3 Parity 365 0 9 2.1 1.5 Length of Stay (days) 372 1 62 4.7 4.4 Length of Surgery (minutes) 369 38 390 104.4 46.4 The majority of hysterectomies were AH (78%), 14% were VH, 5.9% were LAVH and 2.2% were VH converted to AH. Total hysterectomies accounted for 79.8% of hysterectomies, 16.1% were subtotal, and 4% were radical or modified radical hysterectomies. There were no significant differences between patients who had a subtotal and those that had a total hysterectomy for BMI, age, LOS, length of surgery, number of infections, or number of complications. The patients differed only in terms of parity, in that those who underwent a total hysterectomy had more children (2.12 versus 1.66, p = 0.026). A concurrent procedure was performed in 26.6% of patients. This included biopsies (10.5%), reparative surgery (5.9%), procedures to establish urinary continence (3.5%), appendectomies (1.9%), and surgery to manage intra-operative events (2.2%). Table 2 outlines the indications for surgery overall and by type of hysterectomy. There were 526 indications listed for the 372 patients, as up to three reasons could be cited. For 245 of the women (65.9%), only one reason was identified, while 100 women (26.9%) had two reasons and an additional 27 (7.3%) had three reasons listed. Dysfunctional or abnormal uterine bleeding was the most common indication, at 26.4% of the sample. However, this indication accounted for 52.5% of the vaginal hysterectomies, while another 25.4% of the vaginal hysterectomies were for pelvic organ prolapse of stress incontinence. Significance testing of the indications by type of surgery was not carried out due to the large number of cells with a frequency of five or less. Table 2 Indications for surgery by type of hysterectomy. Indication Abdominal Vaginal Lap-Assisted Vaginal Vaginal Converted to Abdominal Total (Row Percent) Percent of Overall Total Dysfunctional or Abnormal Uterine Bleeding 95 (68.3) 31 (22.3) 7 (5.1) 6 (4.3) 139 (100) 26.4% Leiomyomas 80 (95.2) 3 (3.6) 0 1 (1.2) 84 (100) 16.0% Adnexal or Pelvic Mass, Ovarian Neoplasm or Cyst 60 (100) 0 0 0 60 (100) 11.4% Endometrial, Ovarian or Cervical Cancer 54 (93.1) 3 (5.2) 0 1 (1.7) 58 (100) 11.0% Chronic Pelvic Pain, Severe Menstrual Related Mood Disorder or Dysmenorrhea 38 (67.9) 4 (7.1) 13 (23.2) 1 (1.8) 56 (100) 10.6% Endometrial Hyperplasia, Cervical Dysplasia, or Family or Personal History of Cancer 37 (78.7) 3 (6.4) 6 (12.8) 1 (2.1) 47 (100) 8.9% Pelvic Organ Prolapse or Genuine Stress Incontinence 22 (55.0) 15 (37.5) 2 (5.0) 1 (2.5) 40 (100) 7.6% Endometriosis or Adenomyosis 22 (81.5) 0 4 (14.8) 1 (3.7) 27 (100) 5.1% Chronic Salpingitis, Oophoritis, Hydrosalpinx, Pyosalpinx, Post Menopausal Bleed or Other 15 (100) 0 0 0 15 (100) 2.9% Total 423 (80.4) 59 (11.2) 32 (6.1) 12 (2.3) 526 (100) 100% Values are given as N (% of row total), with the exception of the final column, which contains the percentage of the overall total. Note that up to three indications could be listed, resulting in 526 reasons for 372 patients. Fifty-eight (15.6%) of the patients had a diagnosis of cancer pre-operatively, which rose to 76 (20.4%) post-operatively. The population with cancer was older, had higher BMIs, longer surgeries, and longer lengths of stay than those without cancer (Table 3 ). Table 3 Characteristics of patients with and without cancer. Characteristic Cancer N Mean Std. Deviation p-value* BMI No 285 28.0 6.1 .042 Yes 72 30.7 10.7 Age in years No 296 46.7 10.1 < .001 Yes 76 55.9 13.9 Length of Surgery in minutes No 293 98.9 41.1 < .001 Yes 76 125.7 58.4 Length of Stay in days No 296 4.3 3.4 .022 Yes 76 6.2 7.0 * p-values are based on the two-sample t-test BMI was missing for 4 patients with cancer and 11 patients without cancer; length of surgery in minutes was missing for 3 patients without cancer. Twenty-six patients visited the emergency room within 30 days of their discharge and an additional nineteen patients were readmitted to the hospital. Table 4 compares the characteristics of patients who were not readmitted to those who were seen in the ER or readmitted to the hospital. Table 4 Characteristics of patients readmitted to the ER or hospital. Characteristic Readmission N Mean Std. Deviation p-value* BMI None 303 28.4 6.7 .007 ER Only 25 27.1 6.9 Readmitted 18 33.6 13.4 Age in years None 316 48.9 11.5 .110 ER Only 26 44.0 9.9 Readmitted 19 47.6 12.7 Length of Stay in days None 316 4.6 3.4 .007 ER Only 26 3.8 1.5 Readmitted 19 7.7 13.4 * p-values are based on one-way analysis of variance Infections occurred in 15.3% of patients, including urinary tract infections (7.5%), incision site infections (5.6%) and pelvic infections (2.2%). Those who developed an infection had a higher mean BMI (p = 0.018), longer LOS (p = 0.018) and longer length of surgery (p = 0.036) than those who did not. Four percent of patients had a repeat laparotomy or unplanned laparotomy (not including those for conversion of VH to AH). Other complications occurred in 24.5% of patients, the most common being excessive bleeding (11.3%) and post-operative ileus (5.4%). Other complications involving the bladder, bowel, pulmonary function, cardiac function or drug reactions occurred in less than 2% of patients respectively. Table 5 contains the characteristics of the women by oophorectomy and hysterectomy type (excluding LAVH and VH converted to AH). Overall, 65% of women had both or last ovary removed, including 57% of the 257 premenopausal women and 84% of the 113 postmenopausal women (menopausal status was not documented in two patients). In women with dysfunctional uterine bleeding as the only indication, 35% had both or last ovary removed. In women with leiomyomas as the only indication, 71.4% had both or last ovary removed. Table 5 Characteristics of Women By Oophorectomy and Hysterectomy Categories Characteristic Mean (SD) Oophorectomy Hysterectomy No Ovaries Removed n = 129 Both or Last Ovary Removed n = 243 Abdominal n = 275 Vaginal n = 52 Age in Years 44.3 (10.7) 50.8 (11.4)* 49.4 (11.5) 47.4 (11.9) Body Mass Index 27.4 (5.3) 29.2 (8.2)* 29.2 (7.8) 25.8 (4.6)† Length of Stay in Days 3.8 (1.7) 5.2 (5.2)* 5.2 (4.8) 3.0 (1.6)† Length of Surgery in Minutes 109.3 (56.8) 101.8 (39.8) 106.3 (48.7) 84.7 (34.6)† * Between-group differences significant at p < .05, 2-sample t-test † Between-group differences significant at p < .01, 2-sample t-test A comparison of the abdominal and vaginal approaches revealed no differences in terms of incidence of infection, readmission to the ER or hospital, incidence of excessive bleeding or complication rate. LAVH and VH converted to AH were excluded from all regression analyses as they represented subgroups that were clinically different than routine AH and VH. Table 6 presents the results of the linear regression modeling for length of surgery and LOS. All variables with a significance level of p < .20 in the bivariate analyses were offered into the models. Predictors of length of surgery included higher BMI, younger age, higher parity, a higher number of concurrent procedures and an abdominal approach. These predictors account for 33.1% of the variation in length of surgery. Predictors of a longer LOS include an abdominal approach, older age and a higher number of concurrent procedures. Oophorectomy, which was significantly associated with LOS in the bivariate analyses, was not retained in the model since it was also associated with the abdominal approach, resulting in collinearity between the two variables. In order to normalize the distribution, the LOS regression model was developed without two outliers that had LOS of 45 and 62 days. The three predictors accounted for 19% of the variation in LOS. Post-hoc analyses (scatter plots of the residuals against the predicted values, influence diagnostics) were done to examine the model fitting and indicated that the fit was acceptable. Table 6 Predictors of Length of Surgery and Length of Stay based on Linear Regression Length of Surgery in minutes (r 2 = .331) Coefficient p-value Constant 77.11 BMI 1.16 < .001 Age in years -0.55 .008 Parity 4.06 .009 Number of concurrent procedures 45.24 < .001 Vaginal approach (compared to abdominal) -22.56 < .001 Length of Stay in Days (r 2 = .189) Constant 2.3 Vaginal approach (compared to abdominal) -1.7 < .001 Age in years 0.043 < .001 Number of concurrent procedures 1.2 < .001 Additional variables offered into the Length of Surgery model (but not selected) included menopausal status, number of indications, cancer as primary indication and oophorectomy. Additional variables offered into the Length of Stay model included BMI, cancer as primary indication and oophorectomy. Logistic regression for approach of hysterectomy indicated that a patient was 1.1 times more likely to have an AH for each one-point increase in BMI (p = 0.003), 47.6 times more likely to have an AH if she had a concurrent unilateral or bilateral oophorectomy (p < 0.001) and 1.7 times more likely to have a VH with each additional child (p < 0.001). Discussion The majority of the patients were overweight (29.6%, BMI 25–29.9) or obese (36.6%, BMI ≥ 30). These numbers define a population whose obesity level is 21.8 percentage points above the national average and although there is no known average BMI for all hysterectomy patients in Canada for comparison, the high obesity rate at this centre may have contributed to the reliance on the abdominal approach. A patient was in fact eleven times more likely to have an AH for every 10-point increase in BMI. Although recent studies exclude BMI as a factor in determining the route of hysterectomy, it has been noted that obesity of the buttocks may interfere with the exposure necessary for a VH [ 5 ]. The general trend in determining the route of hysterectomy has been to challenge the validity of the exclusionary criteria for VH, such as nulliparity, larger uterine size, previous cesarean delivery, and pelvic laparotomy. These are no longer considered to be strong contraindications to a vaginal approach [ 5 - 11 ]. Yet the abdominal approach is still the most utilized approach at this facility, accounting for 78% of the hysterectomies. The general impression from this and other studies is that surgeon expertise, patient weight and the need for adnexal surgery may play the strongest roles in determining the ultimate route for hysterectomy [ 6 - 12 ]. The need for concurrent oophorectomy may also have been a contributing factor. Oophorectomies, while able to be performed vaginally in the majority of circumstances, were more likely to have been performed abdominally in this population due to issues of accessibility (size of patient). The overall ratio of abdominal to vaginal (alone or in conjunction with laparoscopy) surgeries is 5.6:1 but when only considering those surgeries performed for indications other than cancer (cancer found pre or post operatively), the ratio reduces to 3.9:1. This is consistent with the fact that most malignant indications for surgery require an abdominal approach in order to ensure access to structures and to allow for staging procedures. Our data did not demonstrate a significant difference between AH and VH in terms of outcome variables such as the rate of infection or complication, however, the two day reduction in LOS for VH may have significant cost reduction potential [ 8 , 13 ]. In our study, less than 20% of the hysterectomies performed in 2001 were VH or LAVH. This is below the average rate of 32% across Canada for 1999–2000 [ 14 ]. The average length of stay for hysterectomy was 4.7 days, which is only slightly above the average Canadian value of 4.4 from 1999–2000 [ 14 ]. In light of this comparison, an effort to increase the proportion of hysterectomies performed using a vaginal approach would be in keeping with the Society of Obstetricians and Gynecologists of Canada clinical practice guidelines which recommend offering VH to all women where that approach is deemed feasible by the surgeon [ 15 ]. Recent reports [ 16 ] have demonstrated a marked improvement in the ratio of VH to AH with the adoption of guidelines that clearly determine the correct surgical approach based on vaginal access, mobility with the Valsalva maneuver and uterine size. The application of guidelines [ 17 ] such as these warrants careful consideration in centers where a mandate exists to increase the rate of VH. The merit of performing a concurrent oophorectomy during hysterectomy continues to be debated for women not at high risk of developing ovarian cancer. Estimates regarding the number of prophylactic oophorectomies needed to prevent one case of ovarian cancer range from 200 [ 18 ] to 300 [ 19 ]. Benefits such as prevention of ovarian cancer and perhaps breast cancer have to be weighed against an instantaneous surgical menopause that may increase a woman's risk of ischemic heart disease and osteoporosis [ 18 ]. In addition, although not all women decide to take HRT after oophorectomy, those that do, have to additionally consider the risks and benefits associated with that treatment. The main outcome from a recent study that investigated women's attitudes towards oophorectomy as an adjunct to hysterectomy concluded that while over half the women expressed a desire to decline oophorectomy, the majority were not well informed as to the long-term consequences of either decision [ 20 ]. Few clear guidelines exist to aid either the physician or the patient in the decision making process, making it all the more important to ensure that the patient is adequately informed about the long and short term risks and benefits of all treatment options. The limitations of this study include uneven distribution of patients in each treatment group and lack of randomization due to the nature of the retrospective chart review process. Furthermore, because the audit process relied entirely on chart documentation, information may have been missed or incorrect as a result of improper or absent documentation. The broad range of information collected also prevented the researchers from employing more rigorous definitions and verification of outcomes. The retrospective nature of the study precluded an evaluation of the decision making process leading to oophorectomy as well as the influence of pre-operative indications, uterine size, parity, previous c-section and concurrent oophorectomy on surgical approach. This would need to be addressed prospectively, by surveying the surgeons at the time that the decision was made. Conclusions Both the patient and the health care system may benefit from the trend towards increased use of vaginal hysterectomies. However, the abdominal approach continues to dominate, likely related to patient size, surgeon preference and the need for adnexal surgery. The audit process proved to be an important method by which to assess trends associated with common surgical procedures. This study raises important questions about the relationship between patient characteristics, surgical approach and the indications for surgery, and a prospective approach, designed to address these questions more fully, is now indicated. Furthermore, in light of recent evidence [ 16 ] demonstrating the impact of a directed approach to affect the ratio of AH to VH, clear guidelines as provided by the Society of Pelvic Reconstructive Surgeons [ 17 ] should be considered to invariably increase the rate of VH. This study raises important questions about the relationship between patient characteristics, surgical approach and the indications for surgery, and a prospective approach, designed to address these questions more fully, is now indicated. Competing Interests The author(s) declare that they have no competing interests. Authors' Contributions AT performed data collection, participated in the study design and coordination and participated in drafting the manuscript. WH performed the statistical analysis and participated in drafting the manuscript. RHG conceived the study and participated in its design and coordination and participated in drafting the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535934.xml
514911	Individualizing therapy – in search of approaches to maximize the benefit of drug treatment (II)	Adjusting drug therapy to the individual, a common approach in clinical practice, has evolved from 1) dose adjustments based on clinical effects to 2) dose adjustments made in response to drug levels and, more recently, to 3) dose adjustments based on deoxyribonucleic acid (DNA) sequencing of drug-metabolizing enzyme genes, suggesting a slow drug metabolism phenotype. This development dates back to the middle of the 20 th century, when several different drugs were administered on the basis of individual plasma concentration measurements. Genetic control of drug metabolism was well established by the 1960s, and pharmakokinetic-based individualized therapy was in use by 1973.	Patterns of drug prescription Despite tremendous advances in the science and technology of drug development, as well the emergence of guidance and consensus building among scientists, many clinicians, pharmacists, and consumers remain uninformed regarding the scientific basis of establishing bioequivalence, the generic-drug approval process, and the issues related to individualizing therapy in general [ 7 , 8 ]. The consequence may be drug dosing errors: overdosing or underdosing of drugs, resulting in the occurrence of harmful effects or the nonoccurrence of the expected treatment benefit. Recent information [ 9 ] indicates that doctors are not consistently prescribing proven treatments at recommended doses, and at times they are not prescribing proven treatments at all. A decrease in dose may decrease the efficacy ( relative risk reduction [RRR]) of therapy and thereby decrease the treatment's net benefit . Not prescribing an agent will effectively nullify the potential benefit to individuals, and when repeated frequently enough, failure to prescribe the agent will significantly decrease the benefit to the population as a whole. At other times, doctors tend to prescribe a drug more generally than clinical trials dictate. The treatment of a population with lower outcome prevalence (OP) decreases net benefit and may lead to harm . Overdosing may increase treatment-related harm, and underdosing may erode efficacy; both will result in diminished treatment benefits. Finally, noncompliance on the part of the patient may lead to a decrease in efficacy and a requisite decrease in net treatment benefit. If a patient reduces the dose without totally eliminating the drug, the risk of non-dose-related side effects of treatment may remain. The relationship between the terms mentioned above, which govern treatment success, can be expressed mathematically as follows [ 10 - 12 ]: Net Benefit = RRR * OP - Harm The graphical representation in Figure 1 allows for a series of observations that expand our understanding of the benefits and risks of treatment. Figure 1 Basic relationships. Net benefit is plotted as a function of outcome prevalence. The line represents the relationship with the assumption of a relative risk reduction of 30% and treatment harm of 0%. The line is the maximum net benefit that can be attained at any given outcome prevalence. The x-intercept , or benefit threshold, represents the outcome prevalence at which net benefit will accrue to individuals and the population as a whole. The point of maximum benefit occurs when the outcome prevalence is 100%; at this point, if harm is absent, the net benefit or efficiency of treatment equals the relative risk reduction or efficacy of that treatment. Benefit decreases proportionately as a function of outcome prevalence. The difficulties in drug prescription that are mentioned above are not related to the physicians' training or experience but result instead from difficulties in relying with confidence on label claims of efficacy, safety, and interchangeable use of new drugs "within class." The common denominator among these problems seems to be an insufficiently well crystallized knowledge base regarding the proper use and interpretation of general terms such as equivalence/similarity, pharmaceutical equivalence/therapeutic equivalence , and bioequivalence/bioavailability . Problems are further compounded by the increasing use of generic drugs and the interpretation of such terms as prescribability and switchability . Term definitions Bioavailability (BA) indicates a measurement of the rate and amount of therapeutically active drug that reaches the general circulation and its presumed site of action [ 13 ]. Bioequivalent drug products (BE) Bioequivalence is the absence of a significant difference in the rate at which, and the extent to which, the active ingredients in pharmaceutical equivalents become available at the site of drug action in the body when administered under similar experimental conditions in an appropriately designed study. A product may also be considered bioequivalent to an innovator product if (a) the difference in rate of drug absorption between the two products is intentional and (b) no significant difference is found in the extent of absorption of the two products when they are evaluated under similar experimental conditions [ 13 - 17 ]. Bioequivalence requirement refers to a requirement, imposed by the Food and Drug Administration (FDA), of in vitro and/or in vivo testing of specified drug products that must be satisfied as a condition of marketing [ 17 ]. Pharmaceutical alternatives are drug products that contain the identical therapeutic moiety, or its precursor, but not necessarily in the same amount or dosage form as the same salt or ester. Each such drug product individually meets either the identical or its own respective compendial or other applicable standards of identity, strength, quality, and purity, including potency and, where applicable, content uniformity, disintegration times, and/or dissolution rates [ 17 ]. Pharmaceutical equivalence To be considered pharmaceutically equivalent, two drug products must (a) contain identical amounts of the same active ingredients in the same dosage form, (b) be formulated to meet the same compendial or other applicable standards of quality and purity, and (c) generally be labeled for the same indications. However, pharmaceutical equivalents may differ in the excipients (e.g., flavors, preservatives) that they contain, as well as in their shape, scoring, packaging, and in certain circumstances, their labeling [ 17 ]. Average bioequivalence involves assessment of pharmacokinetic parameters such as area under the curve (AUC) and peak concentration (Cmax), as well as calculation of a 90% confidence interval for the ratio of the averages of these parameters for the two products that are compared, usually a test product (T) against a reference product (R). The calculated confidence interval should fall within a conventionally established BE limit of 80% to 125% for the ratio of the product averages [ 18 ]. The clinical judgment underlying this BE limit is that a test product with BA measures outside this range would be denied market access. However, in specified circumstances, clinical judgment permits widening or narrowing of the BE limit (e.g., 90% to 111% for narrow-therapeutic-range drugs and drug products). Since the implementation of the FDA Bioavailability and Bioequivalence Requirements in 1977, the assessment of bioequivalence has been a subject of continuous debate [ 16 , 20 - 22 ]. This controversy has led to modifications in the bioavailability/bioequivalence regulations and guidelines. Despite these modifications, the process of assessing bioequivalence continues to evolve as scientific consensus emerges on many of the issues driving the debate [ 23 ]. The average BE reflects comparison of population averages and therefore fails to assess the subject-by-formulation interaction variance (i.e., the variation of the averages in particular individuals). In contrast, the newer population and individual approaches reflect differences in the objectives of BE testing at various stages of drug development. These differences are embodied in the concepts of prescribability and switchability (interchangeability) [ 19 - 22 ]. These concepts underscore the difference between the population and individual bioequivalence approaches. Population bioequivalence assesses total variability of the measure in the population, and it becomes important when physicians are initially prescribing a medication and they need to rely on the average performance of the drug product [ 24 ]. In contrast, the most important consideration for individual bioequivalence rests on the assurance that products deemed bioequivalent can be used interchangeably in the target population (i.e., they exhibit switchability) [ 25 ]. In addition to the comparison of averages, the individual bioequivalence approach compares within-subject variabilities and assesses subject-by-formulation interaction. It offers flexible equivalence criteria based on the individual therapeutic window and variability of the reference drug product. Furthermore, it allows scaling criteria for highly variable/narrow-therapeutic-range drugs and promotes the use of subjects from the general population in bioequivalence studies. Prescribability refers to the clinical setting in which a practitioner prescribes a drug product to a patient for the first time. In this setting, the prescriber relies on the understanding that the average performance of the drug product has been well characterized and relates in some definable way to the safety and efficacy information from clinical trials. Switchability refers to the setting in which a practitioner transfers a patient from one drug product to another. This situation arises with generic substitution, as well as with postapproval changes by an innovator or a generic firm in the formulation and/or manufacture of a drug product. Under these circumstances, both the prescriber of the drug and the patient should be assured that the newly administered drug product will yield safety and efficacy comparable to that of the product for which it is being substituted. However, such a switch may, in fact, occur without the patient's or clinician's knowledge, and this concern is addressed in equivalence studies designed to minimize the risk to the patient in both situations. Although average bioequivalence is the recommended parameter for most bioequivalence studies, the current FDA guide titled Statistical Approaches to Establishing Bioequivalence [ 26 ] recommends that population and individual bioequivalence also be evaluated in some cases. Understanding of this process is enhanced by the outline in Figure 2 , which illustrates the classical exposure-response relationship that might assist in adjusting dosages and dosing regimens in the presence of influences on pharmacokinetics (PK) by demographic factors (e.g., age, gender), intrinsic factors (e.g., impaired organ function), or extrinsic factors (e.g., concomitant medication, food intake). Figure 2 Exposure-response relationships. Relationships between (1) drug substance and drug product, (2) exposure expressed as dose or systemic exposure on log scale, and (3) positive (efficacy) or negative (toxicity) outcomes. These outcomes may be measured by clinical end points, surrogate endpoints, or biomarkers. The relationships between exposure and outcomes define the optimal dose and therapeutic window. The term change introduces the concept of equivalence in outcomes before or after a specified change (eg, generic substitution, postapproval manufacturing change). CMC, Chemistry, manufacturing, and controls; BA/BE, bioavailability and bioequivalence; PK, pharmacokinetics; PD, pharmacodynamics. The outcomes (clinical benefit, reflecting the "response" component of the relationship), can be measured as clinical or surrogate endpoints or as biomarkers. The relationship between exposure and outcomes, expressed as dose- or concentration-response curves that have efficacy and toxicity levels at their extremes, define the optimal dose and the therapeutic window [ 27 ]. Theoretically, these curves should be generated in different individuals for developing prescribability criteria, and in the same individuals for developing switchabiliy criteria. Premarketing risk-benefit assessment The approach described above is a simplified framework of successive steps to be taken in phases I and II of the development of any drug, with the aim of generating data for drug labeling (i.e., data on dose-response [effectiveness and toxicity] relations of the new agent and how these depend on patient characteristics). The dose regimen of such a new agent, explored and established through phases I and II, is to be demonstrated in phase III as "safe and effective" for the claimed indication. Proper study design and research methodology, as well as appropriate statistical analysis, should be applied to ensure that the drug's effectiveness can be substantiated. Further, the outcome derived from two such randomized clinical trials should document that the estimate of the true treatment effect favors the new drug over the reference drug and that the toxicity of the new agent does not exceed acceptable limits. In other words, a first risk/benefit assessment would favor the new drug. Having come that far, a question justifiably arises: what logic leads to the conclusion that the new drug is likely to be effective in future patients? That is, are the treatment benefits generalizable to an actual clinical population? The question is extended to external validity , meaning the extent to which the conclusions of a study would hold true for other persons, in other places and at other times. Examples from most therapeutic areas indicate that approved drugs may not work as expected when applied in broad community populations, in real-world settings, and among diverse practitioners operating under real-world constraints [ 28 ]. In such instances, years of effort and huge investments fail to meet the intent of providers, the expectations of consumers, or the demands of healthcare payers. Conventionally, the dose-response trials mentioned above, exploring the behavior of biomarkers both cross-sectionally between individuals treated with different doses of an agent, and longitudinally within individuals as doses (or concentrations) change with time, supply the necessary information on the drug's pharmacologic action (the so-called empiric confirmation at a conventional α level). However, despite all their methodological rigorousness, these trials may not entirely eliminate factors that influence bioavailability in earlier development stages and internal validity in later-phase studies (randomized controlled trials [RCTs]). Bioavailability may be influenced by • Patient-related factors, such as concurrent diseases, differences in first-pass metabolism, interactions with concomitant medications, diet, circadian biorhythms, the influence of fed-versus fasted-state physiologic conditions, and gastrointestinal factors (e.g., pH, motility, blood flow, bacterial flora) [ 29 - 32 ]. • Product-related factors, such as: physical and chemical properties of the drug (e.g., solubility, degree of ionization, crystalline forms, chemical form, isomers), as well as variables related to manufacturing, formulation, or both (e.g., coatings, compression force, particle size, presence or absence of excipients) [ 30 - 33 ]. To control for as many variables as possible, most bioequivalence trials are conducted with healthy volunteers as subjects, [ 19 , 34 , 35 ] and in real patients only in circumstances wherein the use of volunteers would be unethical (e.g., tests with cytotoxic drugs) or when assessment of bioequivalence is based on pharmacodynamic and/or clinical end points [ 35 ]. The causal confirmation is much more complex than the empiric confirmation, even if the drug's previously established pharmacologic action is believed to ensure that the drug has the same intrinsic property that alters the clinical outcome – in the RCT patients – in a similar way and to a similar extent as in the previous patients. A number of other causes, however, may interfere in the conduct phase of an RCT. These causes include • Confounding – a distortion of an association between an exposure and disease brought about by extraneous factors. • Interaction – the interdependent operation of two or more factors that produce an unanticipated effect. • Transience – an idiosyncratic property of a drug that displays its expected pharmacologic property when tested in one batch, but not in others. Many of the specific design and analysis features applied to RCTs (e.g., blinding, randomization, intention-to-treat analysis) are meant to ensure that the possibility of confounding is minimized or eliminated. The same does not apply in the case of transience or interaction, for which independent evidence is needed to eliminate those possibilities. Highly variable drugs Drugs that tend to exhibit high degrees of variability in their pharmacokinetic profiles are known to complicate the assessment of bioequivalence [ 22 , 36 , 37 ]. This is, at least in part, a function of the high intrasubject variability (previously defined as greater than 30%) of the drug or drug product [ 22 ]. Examples of such drugs are propafenone immediate release, verapamil, and nadolol. Narrow-therapeutic-index drugs Small changes in systemic concentration of such drugs can lead to marked changes in pharmacodynamic responses [ 19 - 22 ]. A broader term for the narrow-therapeutic-index drugs is "critical-dose drugs." Characteristically, these agents require blood-level monitoring, need to be dosed on the basis of body weight or other individualized parameters, display serious clinical consequences if overdosing or underdosing occurs, and manifest a steep dose-response relationship [ 38 ]. A typical drug in this category is warfarin, which is widely used for its anticoagulant properties. For the most part, metabolism of S-warfarin occurs by means of the gene CYP2C9 [ 39 ]. Inhibition of this isoform results in several clinically important drug interactions. Fluconazole, metronidazole, miconazole, and amiodarone are a few examples of the many drugs that profoundly inhibit S-warfarin metabolism and produce marked increases in prothrombin time (PT) measurements [ 40 - 43 ]. A multitude of endogenous and exogenous factors may contribute alone or in combination to either increasing or decreasing PT ratio or the INR response [ 44 ]. Factors that increase PT ratio or international normalized ratio (INR) response include: Endogenous factors: 11 Exogenous factors: 117 specific drugs and 49 different classes of drugs Factors that decrease PT ratio or INR response include: Endogenous factors: 5 Exogenous factors: 42 specific drugs and 24 different classes of drugs Physician surveys have indicated that most clinicians favor more rigid bioequivalence guidelines for these types of drugs [ 8 ]. Others have recommended that the bioequivalence requirement for these agents be based on intrasubject variability, as well as the pharmacokinetic-pharmacodynamic relationship. Although the FDA has not modified the bioequivalence guidelines for critical-dose drugs, the Canadian regulatory authority, Health Canada, has narrowed the CI requirement for these drugs to 90% to 110%. For prescribability, the current requirements may be adequate for all drugs, including those with a narrow therapeutic index. However, some clinicians have expressed concerns about switchability [ 7 , 39 ]. Several reports suggest that once a patient has been carefully titrated on a narrow-therapeutic-index drug, the formulation should not be switched [ 7 , 8 ]. The same might be true of warfarin. Drug products suspected of having bioequivalence problems are listed in the Orange Book (Approved Drug Products with Therapeutic Equivalence Evaluations) [ 45 ]. Such drugs may exhibit a narrow therapeutic index or solubility problems, or they may be poorly absorbed or unstable in gastrointestinal fluids [ 33 ]. Among clinicians there seems to be widespread concern that even small changes in the bioavailability of drugs' active ingredients might lead to significant changes in the efficacy or safety of those products [ 7 , 8 , 38 ]. The concept of risk and its application to drug development In light of serious concerns about risks incurred from using medical products, a variety of public and private agencies involved in health care are dedicating more attention to examining the current system of managing these risks. The main goal is to focus on the costs and value of better data concerning the incidence and causes of injuries from medical products and the roles of all stakeholders in risk management. The FDA has issued a concept paper [ 46 ] presenting preliminary thoughts on risk issues, including: • Important risk assessment concepts. • Generation and acquisition of safety data during product development. • Analysis and presentation of safety data in an application for approval. Risk assessment , defined as the process of identifying, estimating, and evaluating the nature and severity of risks associated with a product, should be continuous throughout the life cycle of any product, whereas the process of risk management intervention is intended to enhance the safe use of a product by reducing risk. With regard to the current trend toward systemic risk confrontation , the FDA, Center for Drug Evaluation and Research (CDER), and Center for Biologics Evaluation and Research (CBER) appear ready to develop and finalize, by fall 2004, guidance documents regarding risk assessment, clinical pharmacovigilance, and risk management. Table 1 highlights the components of a risk management system (RMS). Table 1 Components of a Risk Management System • Risk Assessment – estimation & evaluation of risk • Risk Confrontation – determination of acceptable levels of risk in societal context • Risk Intervention – actions to control risk • Risk communication – interactive process of exchanging risk information • Risk Management Evaluation – measurement of effectiveness of aforementioned activities Conclusions Determining the optimal initial dosage regimen (prescribability) and maintaining safety and efficacy outcomes when the regimen is changed in some way (switchability) demand careful decision making in the application of equivalence approaches. These approaches must be applied differently during the three phases of the drug development process, and the knowledge-base that is derived from this process must be transferred to and utilized by physicians and pharmacists to assist them in prescribing and dispensing medicines to patients. Consistent and appropriate management of equivalence approaches supports good assessment, management, and communication about risks associated with a therapeutic product, as expressed in product labeling, as well as in specifications and standards that control the quality of a therapeutic product in the marketplace.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514911.xml
261870	Viral Discovery and Sequence Recovery Using DNA Microarrays	Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.	Introduction Over the past two decades, technological advances in molecular biology have fuelled progress in the discovery of new pathogens associated with human diseases. The identification of novel viruses such as hepatitis C virus ( Choo et al. 1989 ), sin nombre virus ( Nichol et al. 1993 ), and Kaposi's sarcoma herpesvirus ( Chang et al. 1994 ) has relied upon a diverse range of modern molecular methods such as immunoscreening of cDNA libraries, degenerate PCR, and representational difference analysis, respectively. In spite of these successes, there remain numerous syndromes with suspected infectious etiologies that continue to escape identification efforts, in part due to limitations of existing methodologies for viral discovery ( Muerhoff et al. 1997 ; Kellam 1998 ). These limitations, coupled with the constant threat posed by newly emerging infectious diseases of unknown origin, necessitate that new approaches be developed to augment the repertoire of available tools for pathogen discovery. We have previously described a prototype DNA microarray designed for highly parallel viral detection with the potential to detect novel members of known viral families ( Wang et al. 2002 ). This microarray contained approximately 1600 oligonucleotides representing 140 viruses. Building upon this foundation, a more comprehensive second-generation DNA microarray consisting of 70mer oligonucleotides derived from every fully sequenced reference viral genome in GenBank (as of August 15, 2002) was constructed. The most highly conserved 70mers from each virus were selected as described by Wang et al. (2002 ) to maximize the probability of detecting unknown and unsequenced members of existing families by cross-hybridization to these array elements. On average, ten 70mers were selected for each virus, totaling approximately 10,000 oligonucleotides from approximately 1,000 viruses. The objective was to create a microarray with the capability of detecting the widest possible range of both known and unknown viruses. This pan-viral microarray was used as part of the global effort to identify a novel virus associated with severe acute respiratory syndrome (SARS) in March 2003, as reported by Ksiazek et al. (2003 ). We describe here the experimental details of the microarray methodology for novel virus identification, using the SARS outbreak as an example. Results During the initial phase of research into the etiology of SARS, an unknown virus was cultured in Vero cells from a patient suffering from SARS ( Ksiazek et al. 2003 ). Total nucleic acid purified from this viral culture, as well as a control culture, was obtained from the Centers for Disease Control and Prevention on March 22, 2003. These two samples, along with additional controls (HeLa cell RNA and water alone), were amplified and hybridized within 24 h to the virus DNA microarray. The strongest hybridizing array elements from the infected culture were derived from two families: astroviridae and coronaviridae. Table 1 lists the oligonucleotides from these families with the greatest hybridization intensity. By comparison, these oligonucleotides yielded essentially background levels of hybridization in the various control arrays performed in parallel. The initial suggestion from this hybridization pattern was that members of both of these viral families might be present. However, alignment of the oligonucleotides using ClustalX revealed that all four hybridizing oligonucleotides from the astroviridae and one oligonucleotide from avian infectious bronchitis virus (IBV) (GenBank NC_001451), an avian coronavirus, shared a core consensus motif spanning 33 nucleotides (data not shown); thus, these five oligonucleotides behaved essentially as multiple redundant probes for the same sequence. This motif is known to be present in the 3′ UTR of all astroviruses and the avian coronaviruses ( Jonassen et al. 1998 ), but appears to be absent in the available sequenced mammalian coronaviruses (bovine coronavirus, murine hepatitis virus [MHV], human coronavirus 229E, porcine epidemic diarrhea virus, and transmissible gastroenteritis virus). The other three hybridizing oligonucleotides were derived from three conserved regions within the ORF1AB polyprotein common to all coronaviruses ( Figure 1 ). Based on the aggregate hybridization pattern, the virus appeared to be a novel member of the coronavirus family. Figure 1 Prototypical Coronavirus Genome Structure Red bars indicate physical location of virus microarray DNA elements mapped to a generic coronavirus genome. Portions of the coronavirus genome sequenced by physical recovery and PCR methods are highlighted with homologies to known coronaviruses. Abbreviations: aa, amino acid; nt, nucleotide Table 1 Oligonucleotides Hybridizing to Viral Sample Underlined nucleotides represent regions of identity to the SARS coronavirus. Does not include reverse complement oligos a BLAST identities to the SARS coronavirus genome (NC_004718) To further characterize this virus, we sequenced fragments of the viral genome using two complementary approaches. First, BLAST alignment of two of the hybridizing viral oligonucleotides, one each from bovine coronavirus and human coronavirus 229E, to the IBV genome indicated that the oligonucleotides possessed homology to distinct conserved regions within the NSP11 gene (BLAST identity matches of 42/47 and 26/27, respectively). A pair of PCR primers was designed to amplify the intervening sequences between the two conserved regions, and a fragment that possessed 89% identity over 37 amino acids to MHV, a murine coronavirus, was obtained ( Figure 1 ; sequence available as Data S1 ). In a parallel approach, we directly recovered hybridized viral sequences from the surface of the microarray. This procedure took advantage of the physical separation achieved during microarray hybridization, which effectively purified the viral nucleic acid from other nucleic acid species present in the sample . Using a tungsten needle, the DNA microarray spot corresponding to the conserved 3′ UTR motif was repeatedly scraped and the hybridized nucleic acid was recovered. This material was subsequently amplified, cloned, and sequenced ( Figure 2 ). The largest clone spanned almost 1.1 kb; this fragment encompassed the 3′ UTR conserved motif and extended into the most 3′ coding region of the viral genome. BLAST analysis revealed 33% identity over 157 amino acids to MHV nucleocapsid, thus confirming the presence of a novel coronavirus (see Figure 1 ; see Data S1 ). We subsequently confirmed results obtained from both strategies described above by using a random-primed RT-PCR shotgun sequencing approach that generated contigs totaling approximately 25 kb of viral genome sequence (see Data S1 ). Figure 2 Viral DNA Recovery and Sequencing Scheme Hybridized viral sequences were physically scraped from a DNA microarray spot, amplified, cloned, and subsequently sequenced. Discussion In this report, we have demonstrated the viability of detecting novel pathogens via cross-hybridization to highly conserved sequence motifs. With the recent sequencing of the complete SARS coronavirus genome (GenBank NC_004718) ( Marra et al. 2003 ; Rota et al. 2003 ), we were able to retrospectively determine the degree of nucleotide identity shared between the hybridizing oligonucleotides and the new coronavirus genome (see Table 1 ). Stretches of relatively uninterrupted nucleotide identity as short as 25 nucleotides yielded clearly detectable hybridization signal, confirming that novel viruses with only limited homology to known viruses can be successfully detected by this strategy. A key feature of this approach is that direct recovery of hybridized material from the microarray provides a rapid route for obtaining sequences of novel viruses. By contrast, conventional strategies for subsequent sequence identification would require time-consuming steps such as library screening or additional rounds of PCR primer design and synthesis. In the case of SARS, we were able to ascertain within 24 h that a novel coronavirus was present in the unknown sample, and partial genome sequences of this virus were obtained over the next few days without the need for specific primer design. To our knowledge, this is the first demonstration of the feasibility and utility of directly recovering nucleic acid sequences from a hybridized DNA microarray. In light of the continuous threat of emerging infectious diseases, this overall approach will greatly facilitate the rapid identification and characterization of novel viruses. Materials and Methods Nucleic acid isolation Total nucleic acid was purified using the automated NucliSens extraction system (BioMerieux, Durham, North Carolina). Following the manufacturer's instructions, 100 μl of each specimen was added to tubes containing 900 μl of prewarmed NucliSens lysis buffer and incubated at 37°C for 30 min with intermittent mixing. Fifty microliters of silica suspension provided in the extraction kit was added to each tube and mixed. The mixtures were then transferred to a nucleic acid extraction cartridge and loaded onto the extractor workstation for processing. Approximately 50 μl of total nucleic acid eluate was recovered. Amplification For the culture supernatants, 450 ng of nucleic acid was used as input for the amplification protocol. In parallel, 50 ng of HeLa cell RNA was used as a positive amplification control and water was used for a negative control. Samples were amplified using a random-primer protocol as described by Wang et al. (2002 ), with the following modifications: first- and second-strand synthesis were primed using primer-A (5′-GTTTCCCAGTCACGATCNNNNNNNNN) followed by PCR amplification using primer-B (5′-GTTTCCCAGTCACGATC) for 40 cycles. Aminoallyl-dUTP was incorporated into the PCR product using an additional 20 cycles of thermocycling. A detailed protocol is available as Protocol S1 . Microarray hybridization and analysis DNA microarrays were printed and hybridized essentially as described by Wang et al. (2002 ), with the following modifications: for array printing, a single-defined 70mer (spike-70) was mixed with each viral oligonucleotide in a 1:50 ratio. Array hybridizations used Cy5-labeled amplified probe from either virally infected cultures or controls (mock-infected culture, HeLa RNA, or water); a reference signal for every spot on each array was generated by using a Cy3-labeled version of the reverse complement of spike-70. Oligonucleotides were assessed by Cy5 intensity. Oligonucleotides from the astrovirus and coronavirus families that passed a conservative, arbitrarily set cutoff of (Cy5 infection -Cy5 mock ) > 1500 intensity units are listed in Table 1 . Additional oligonucleotides from these families and their homology to the SARS coronavirus are listed in Table S1 . Array data has been deposited in the Gene Expression Omnibus (GEO) database (accession number GSE546). A complete list of the viral oligonucleotide sequences on the microarray is also available as Table S2 . Conventional PCR using array element sequences PCR primers were designed by aligning the hybridizing oligonucleotides (Oligo IDs 15081544_766 and 12175745_728) to the IBV genome (Fwd: 5′-TGTTTTGGAATTGTAATGTGGAT; Rev: 5′-TACAAACTACCTCCATTACAGCC) and selecting stretches of near-identity. Primer-B-amplified material was used as the template for 35 cycles of thermocycling using the following program: 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s. Direct sequence recovery from the microarray Amplified viral sequences hybridized to individual microarray spots were recovered by scraping a 100 μm area of the microarray using a tungsten wire probe (Omega Engineering, Inc.) mounted on a micromanipulator while visualized by fluorescence microscopy (Nikon TE300). Recovered material was PCR amplified using primer-B, cloned into pCR2.1TOPO (Invitrogen), and sequenced. A detailed protocol is available as Protocol S2 . Shotgun sequencing Primer-B-amplified nucleic acid (see above) was cloned in pCR2.1TOPO, plated on 2xYT/kan plates, and grown overnight at 37°C. White colonies were picked into 384-well plates containing 2xYT/kan plus 8% glycerol and incubated overnight at 37°C. DNA was purified by magnetic bead isolation. DNA sequencing involved adding 3 μl of water to each bead pellet, followed by 3 μl of Big Dye terminator (v3.1) sequencing cocktail, and incubation for 35 cycles of 95°C for 5 s, 50°C for 5 s, and 60°C for 2 min. Reaction products were ethanol precipitated, resuspended in 25 μl of water, and loaded onto the ABI 3730xl sequencer. The resulting sequence reads were trimmed to remove primer sequences from the RT-PCR step and then assembled by Phrap (P. Green, unpublished data). Resulting contigs were screened by blast to remove any contigs with high human or monkey sequence similarity. The remaining contigs were edited to high quality, making any obvious joins. (Sequences are available as Data S1 .) Supporting Information Data S1 Supporting Data (91.5 KB DOC) Click here for additional data file. Protocol S1 Supporting Protocol (28 KB DOC) Click here for additional data file. Protocol S2 Supporting Protocol (39.5 KB DOC) Click here for additional data file. Table S1 Supporting Table (97 KB DOC) Click here for additional data file. Table S2 Supporting Table (2.2 MB XLS) Click here for additional data file. Accession Numbers The Gene Expression Omnibus accession number for the array sequence is GSE546.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC261870.xml
526784	Hemispheric Asymmetries in Biodiversity—A Serious Matter for Ecology	Although the poles are less diverse than the tropics, this decline shows substantial asymmetries between the hemispheres, suggesting that responses to environmental change may differ substantially in the north and the south.	Penguins have been receiving a lot of bad press lately. They are considered somehow counter, spare, strange. Unlike most plant and animal groups, they do not show a peak of species richness towards the equator and a decline towards the poles. This more conventional spatial pattern is conveniently known as the latitudinal diversity gradient because of the strong covariance of richness and other measures of biodiversity that it describes. It is one of the most venerable, well-documented, and controversial large-scale patterns in macroecology ( Willig et al. 2003 ). Equatorial peaks in species richness have characterised the planet since the Devonian (408–362 million years ago) ( Crame 2001 ) and are typical of a wide range of both terrestrial and marine plants and animals ( Gaston 1996 ; Willig et al. 2003 ). Despite the fact that this pattern has been documented since the late 1700s, sustained interest in both the regularity of the pattern and its likely underlying mechanisms is relatively modern. The realisation that human activity is posing substantial threats to biodiversity has quickened the pace of this interest ( Willig et al. 2003 ). Where the peaks in richness lie (biodiversity hotspots), how these peaks relate to centres of endemism (areas that support large numbers of species that occur nowhere else), and how these patterns are likely to change through time, especially in the face of major environmental change, are major concerns. Without such knowledge, conservation is unlikely to succeed. Although spatial patterns in biodiversity, and particularly the latitudinal gradient, are increasingly well documented for a range of taxa, the proposed mechanisms underlying these gradients remain controversial. In essence, the multitude of mechanisms proposed to explain diversity gradients can be reduced to three categories: historical, ecological, or null. Most significant in raising the temperature of recent discussions is the question of the relative importance of each of these major categories. Historical mechanisms are those that suggest that earth history (e.g., the opening of the Drake Passage and the cooling of Antarctica) and phylogenetic history have played major roles in generating current patterns in diversity, and tend to emphasise regional (and especially longitudinal) differences therein ( Qian and Ricklefs 2004 ; Ricklefs 2004 ). Explanations involving ecological mechanisms often downplay the significance of such regional differences and give most attention to covariation between current diversity and variables such as energy and water availability, and to the ultimate mechanisms underlying this covariation ( Hawkins et al. 2003 ; Currie and Francis 2004 ). By contrast, null models, and specifically the geometric constraints model, argue that the expected pattern of latitudinal variation in richness is not a uniform one, but rather a mid-domain peak, which is almost inevitably the outcome of the random placement of a set of variable species ranges within a bounded domain ( Colwell et al. 2004 ; but see also Zapata et al. 2003 ). It is deviation from the mid-domain expectation that is then argued to be of most interest. In many cases the historical and ecological mechanisms might be difficult to disentangle, such as the historical effects of the establishment of the Antarctic Circumpolar Current, and its consequences for energy availability in the region today ( Clarke 2003 ). Nonetheless, juxtaposing these three major mechanisms raises several questions that could substantially inform the debate in many ways, but have enjoyed far less attention than debating the relative merits of each of them. The geometric constraints model suggests that, to the extent that there is symmetry in the continuity of land (or water) about the equator, declines in richness from the tropical peak should also be symmetrical, with any asymmetries in the latter matching those in the former. Indeed, most texts and reviews dealing with latitudinal diversity gradients only briefly mention hemisphere-related differences and focus instead on the general decline of diversity away from the tropics in both directions (e.g., Brown and Lomolino 1998 ; Willig et al. 2003 ). However, that diversity gradients in the two hemispheres might in many cases be highly asymmetric has long been appreciated ( Gaston 1996 ). Although several historical hypotheses suggest reasons why this asymmetry should exist (reviewed in Brown and Lomolino 1998 ), differences in present ecological factors, such as temperature gradients and rainfall variation, might also explain such asymmetry. If ecological factors are important, then these asymmetries should show up not only in diversity patterns, but also at other levels in the ecological and genealogical hierarchies. From the perspective of ecological explanations for such spatial variation, the questions, then, are how common and strong are such asymmetries, how common are they in patterns of diversity, and what, if any, might be the ecological, rather than null or historical, mechanisms responsible for them? Continents and Climates The last 100 million years have seen both a substantial steepening in latitudinal diversity gradients and the fragmentation of continental land masses ( Crame 2001 ). By 15 million years ago the continents had largely assumed their current positions and a latitudinal temperature gradient very similar to the present one had been established. Today, 70% of all land is in the northern hemisphere, and between latitudes 30° and 60° north, the ratio of water to land is about 1:1, whereas between 30° and 60° south, it is approximately 16:1. The continentality of the north and oceanicity of the south have considerable effects on the climates of the hemispheres, as has long been appreciated ( Bonan 2002 ). Although there is obviously much local and mesoscale variation, terrestrial temperatures in the south (excluding Antarctica) are usually warmer, and much less extreme in terms of their absolute range, than those in the north ( Figure 1A ), especially over the winter months. Southern sites between 30° and 60° typically have mean July temperatures between 0 and 10 °C, whereas at similar latitudes in the north, mean January temperatures vary from −40 to 0 °C. In winter the smaller range of variation in the south is around a physically and biologically significant threshold: the freezing point of water. In the north, winter temperatures are more variable, but generally well below this point. Ocean water temperatures are much less variable than those on land, although variability in the ocean surrounding Antarctica is much reduced compared with that of the Arctic ( Figure 1B ). Figure 1 Temperature Variation with Latitude (A) Mean and absolute minimum and maximum temperatures across the New World.(B) Mean and absolute range in sea surface temperatures across the Pacific at 165° W. Mean annual precipitation is spatially more complex. Overall, precipitation is slightly higher in the south than in the north, at least below 60° latitude. However, much of this precipitation falls over the ocean in the south, leaving the more temperate parts of the southern continents as dry as their northern counterparts ( Bonan 2002 ). Spatial patterns in rainfall variability are also complex, but variability tends to be higher and predictability lower in southern areas. From a biological perspective the significant factor is not necessarily just the magnitude of the variance, but also the mean about which it occurs ( Guernier et al. 2004 ). Clearly, the spatial complexity of climatic variation is much greater than the present overview would suggest. However, these broad brush strokes capture the hemisphere-related variation that might be most significant from a biological perspective. Ecological Consequences If differences in climates do cascade upwards to influence individuals, species, and broader scale patterns in diversity, their influence should be readily detectable at the level of species' life histories and distributions. In birds, large-scale geographic variation in life history variables, such as the incidence of cooperative breeding, extent of parental care, survival, and the timing of reproduction, has been studied for at least the past 50 years, and the mechanisms underlying this variation have been much debated. Taking phylogeny and the idiosyncrasies of the Australian avifauna into account, southern species typically lay small clutches and have long fledging periods, and it is often difficult to predict their date of first laying or, indeed, whether they will lay in a particular year at all ( Covas et al. 1999 ; Russell et al. 2004 ). By contrast, northern species lay larger clutches and have shorter fledging periods, and laying date is more readily predicted, making investigations of phenological shifts associated with modern climatic change more straightforward (e.g., Crick et al. 1997 ). These kinds of differences extend to other taxa. Thus, although the variation of metabolic rate with latitude is becoming increasingly well known for a variety of groups, Lovegrove (2000) has recently suggested, based on comparative work taking both species body mass and phylogeny into account, that unpredictability of resources associated with considerable inter-annual unpredictability in rainfall (in turn partly a consequence of El Niño–associated variability) has been responsible for the evolution of generally low metabolic rates in terrestrial mammals of most of the southern continents. Although El Niño effects are by no means restricted to these regions, it is perhaps low resource availability to start off with, associated with considerable unpredictability, that is of most significance (see also Guernier et al. 2004 ). Insect life histories also show hemisphere-related variation. Low-temperature-related diapause is virtually absent in southern species (e.g., Convey 1996 ), and metabolic rate–temperature relationships are much shallower in the south than the north ( Addo-Bediako et al. 2002 ). The latter is a consequence of relatively cool growing seasons and lack of pronounced seasonality in the south. However, the clearest example of a hemispheric asymmetry is that of cold hardiness strategies ( Sinclair et al. 2003 ). Insects can survive sub-zero temperatures either by tolerating internal ice formation or by reducing their freezing points to avoid ice formation altogether. Although there is further variation within each of these strategies, in general, freeze-avoiding species need to undergo substantial preparation for winter cold and consequently can take some time to emerge from the cold hardy state. This also seems to be true of strongly freeze-tolerant species that can survive freezing far below the point at which they actually freeze. By contrast, moderately freeze-tolerant species—those that can survive only a few degrees of freezing—appear to need little preparation for a freezing event and seem perfectly prepared to continue with their routine activities immediately after thawing. In northern cold climate areas, with the exception of the Arctic, where extremely low temperatures constrain insects to being strongly freeze tolerant, most cold hardy species avoid freezing, whereas in the south most are moderately freeze tolerant ( Figure 2 ). Microclimates reveal why this is the case. As might be expected from macroclimatic variation, southern temperate insects are faced with regular freeze–thaw cycles (i.e., variation about 0 °C), including pronounced summer cold snaps, whereas the continental climates of many areas in the north mean that once temperatures decline below freezing for winter, they stay below this threshold. Figure 2 Latitudinal Variation in Cold Tolerance Strategies in Insects The proportion of insects, as a function of latitude, that are moderately freeze tolerant down to relatively high sub-zero temperatures (moderate FT), that are freeze tolerant down to low sub-zero temperatures (strong FT), that are freeze tolerant but that cannot be classified (other FT), and that are freeze avoiding. North–south asymmetries also show up in snowlines, treelines, the frost tolerance of trees, and the proportion of winter deciduous species ( Woodward 1987 ; Körner 1998 ; Körner and Paulsen 2004 ). Indeed, such differences have long been appreciated for vegetation. In marine systems, one of the best-known asymmetries is the low upper thermal limit to performance and survival in Antarctic compared with Arctic ectotherms. This difference in limits to survival and performance is characteristic of fish, invertebrates, and macroalgae (e.g., Wiencke et al. 1994 ) ( Figure 3 ). Asymmetries are also apparent in the geographic ranges of a wide variety of animals and plants. Rapoport's rule proposes that species ranges will be larger at high than at low latitudes ( Stevens 1989 ). The pattern is thought to be a consequence of considerably greater temporal climatic (and especially temperature) variation at high latitudes, and the resulting need for broader physiological tolerances of individuals. These broad tolerances enable the species to which these individuals belong to occur across a wider range of sites than species at lower latitudes. However, if there is much less temporal temperature variation in the south than in the north, evidence for the rule should be less forthcoming in the southern hemisphere. This is indeed the case. Consistent increases in latitudinal extents with latitude are uncommon in the south, and Rapoport's rule is now largely considered to be a northern phenomenon ( Gaston et al. 1998 ). Figure 3 Variation in Upper Survival Temperatures of Macroalgae from across the Planet Mean and standard error of upper survival temperatures of macroalgae (open symbols, macrothalli; closed symbols, microthalli) from cold areas across the planet. Ant., Antarctic; Arct., Arctic; CT, cool temperate; End., endemic; N, northern hemisphere only; S, southern hemisphere only; N+S, occurrence in both hemispheres. Redrawn from Wiencke et al. (1994) . Large-Scale Asymmetries in Biodiversity In the years since Platnick (1992) suggested that the world is pear-shaped from a biodiversity perspective, with more rapid declines in richness from the equator in the northern than in the southern hemisphere, evidence that there are large-scale asymmetries in the latitudinal diversity gradient has been accumulating. Seed plant and mammalian family richness per unit area declines more steeply in the northern hemisphere than in the south ( Woodward 1987 ; Gaston et al. 1995 ), and similar asymmetries, mostly at the species level, have been noted for other groups such as New World birds, several groups of insects, spiders, foraminiferans, and a variety of benthic marine taxa ( Platnick 1992 ; Rex et al. 1993 ; Eggleton 1994 ; Blackburn and Gaston 1996 ; Culver and Buzas 2000 ; Rodriguero and Gorla 2004 ). Nonetheless, not all groups show these trends, and a recent meta-analysis, albeit one on a relatively coarse scale, failed to find consistent north–south differences in latitudinal gradients ( Hillebrand 2004 ). Recent reviews, particularly of marine diversity, have pointed out the difficulty of making comparisons of this kind owing to sampling constraints ( Clarke and Johnston 2003 ). However, it remains remarkable that even simple exercises—such as plotting richness values for different latitudes or latitudinal bands against each other for the hemispheres and examining the resulting relationship, or overlaying them on the same range of latitudes—rarely appear in the literature. Thus, it is not yet clear how common or strong hemisphere-related asymmetry is. By contrast, it appears that proximate ecological correlates of diversity gradients differ considerably between north and south. Although both historical and ecological factors have led to variation in the numbers and identity of species across the globe ( Ricklefs 2004 ), climate, and particularly energy and water availability, is a strong predictor of broad-scale patterns in species richness for both plants and animals. However, the extent to which energy and water availability constrain species richness varies. In a recent comparative analysis, Hawkins et al. (2003) showed that water availability is the key limiting component of richness for the southern hemisphere, but for temperate regions of the north, energy availability is more important ( Figure 4 ). They ascribe this difference to the warmer and less thermally variable conditions of the southern hemisphere, which, as we have already noted, have considerable effects on species life histories. Figure 4 Latitudinal Variation in the Energy–Water Correlates for Species Richness Latitudinal distribution of energy–water correlates for species richness in which spatial variation in pure energy variables (closed bars), typically measured as temperature or potential evapotranspiration, or spatial variation in pure water availability variables (open bars), typically measured as rainfall or precipitation, best explains richness variation through space. Redrawn from Hawkins et al. (2003) . Of course, biodiversity is not just species richness, but also encompasses the ecological complexes of which species are a part. Although potential north–south asymmetries in interactions have not been widely explored, recent work is providing tantalising glimpses of such variation. Thus, it appears that on the basis of a straightforward (not phylogenetically corrected) comparative analysis, specialisation in plant–pollinator relationships is much greater in the south than in the north. European and North American orchids are typically visited by five species of insects, whereas in southern Africa the median is a single pollinator species per species of orchid ( Johnson and Steiner 2003 ) ( Figure 5 ). Insect–plant interactions might also vary in other ways between the hemispheres, as the rarity of showy autumn colours and the paucity of aphid species—which are thought by some to be a driver of these displays ( Archetti and Brown 2004 )—in south temperate areas suggests. Asymmetries in patterns of human disease point to similar hemisphere-related variation in interactions between organisms ( Guernier et al. 2004 ). Figure 5 Number of Insect Species Pollinating Orchid Species in the Northern and Southern Hemispheres Europe and North America, closed bars, n = 41; southern Africa, open bars, n = 73. Redrawn from Johnson and Steiner (2003) . A World in Flux Despite considerable spatial complexity, there do seem to be regular north–south differences in species life histories and patterns of range size variation that are consistent with disparities in the climates of the two hemispheres ( Figure 6 ). These differences extend to the proximate ecological mechanisms underlying spatial variation in species richness, and, in some cases, apparently to ecological interactions. However, what is less clear is the regularity and strength of north–south differences in spatial diversity patterns, and especially the latitudinal gradient in diversity, as well as the ways in which abiotic variation between the hemispheres might extend through the genealogical and ecological hierarchies to effect such differences. Indeed, if the extent to which abiotic differences between the hemispheres influence biodiversity patterns is to be better comprehended, several key issues deserve attention. Figure 6 Biological Diversity in the Northern and Southern Hemispheres Regular differences between the northern and southern hemispheres in patterns of diversity show up in various groups such as the birds (A) (Adelie Penguin, Pygoscelis adeliae ) and seed plant families (B) (King Protea, Protea cynaroides ). North–south differences in life histories are also apparent in a diverse array of groups ranging from seaweeds (C) (Bull Kelp, Durvillaea antarctica ) and insects (D) (the sub-Antarctic, flightless tineid moth Pringleophaga marioni ) to birds (E) (Cape Sugarbird, Promerops cafer ) and mammals (F) (Sloggett's Rat, Otomoys sloggetti , from the high Drakensberg in South Africa). (Photos: [A, C, and F] Brent J. Sinclair; [B and D] Steven L. Chown; [E] Mhairi L. McFarlane) First, both phylogenetically independent and non-independent comparisons of life history traits and physiological variables across a variety of groups are required. Contrasting these approaches will provide considerable insight into how much of the signal is based on phylogenetic patterns, and how much on current ecological responses. Whilst in some cases data may be obtained from the literature, it is likely that new work will have to be undertaken, especially in the southern hemisphere, where the number of past investigations of such traits is generally much lower than in the north. Moreover, replicated studies using similar methods might substantially improve the signal-to-noise ratio, which can be weakened in “macrophysiological” or large-scale life history and physiological comparisons by the fact that different methods often lead to different outcomes. Second, there is much to be said for the application of similar methods to investigations of large-scale, hemisphere-related patterns of species interactions. Differences like those in plant–pollinator systems discussed here might extend to other interactions in marine and terrestrial systems. Contrasting phylogenetically independent and non-independent comparisons are likely to provide much insight into the reasons for those asymmetries that are found. Finally, comparisons of latitudinal gradients and their underlying correlates in the two hemispheres for the same taxon, sampled using similar methods, and investigated with methods that take cognisance of likely confounding effects are required. This approach will provide a means of determining whether asymmetries in the climates of the two hemispheres really do translate into differences in biodiversity patterns. Such an approach goes to the heart of the question of the processes underlying the latitudinal gradient in species richness, and could go a considerable way to teasing apart the importance of historical, ecological, and null explanations, and identifying the mechanisms that underlie them. In our view, clarifying these issues is of considerable importance. What is at stake is not a set of arcane ecological questions, but rather questions that are central to determining whether ecological and conservation lessons learnt in one area can be applied more broadly. For example, it has been suggested that climate change will cause substantial extinctions in the near future ( Thomas et al. 2004 ). Indeed, responses by species to such change, via phenological shifts and northward movement of species range margins, are well documented for northern hemisphere species ( Parmesan and Yohe 2003 ). However, if there are substantial differences in abiotic environments such that patterns in diversity and their responses to change differ between hemispheres, then such shifts may not be of similar consequence in the south. To date, southern hemisphere studies represent less than 1% of the total in this field ( Root et al. 2003 ), suggesting that it is not at all clear how the considerable biodiversity in the south will respond to future change. We find such a situation extraordinary. Thus, whilst penguins might at first appear counter, spare, and strange, they serve as a reminder that differences between the north and south might not be so much strange, as remarkable and worthy of closer attention.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC526784.xml
523824	The Molecular Basis for Oat Intolerance in Patients with Celiac Disease	ABSTRACT Background Celiac disease is a small intestinal inflammatory disorder characterized by malabsorption, nutrient deficiency, and a range of clinical manifestations. It is caused by an inappropriate immune response to dietary gluten and is treated with a gluten-free diet. Recent feeding studies have indicated oats to be safe for celiac disease patients, and oats are now often included in the celiac disease diet. This study aimed to investigate whether oat intolerance exists in celiac disease and to characterize the cells and processes underlying this intolerance. Methods and Findings We selected for study nine adults with celiac disease who had a history of oats exposure. Four of the patients had clinical symptoms on an oats-containing diet, and three of these four patients had intestinal inflammation typical of celiac disease at the time of oats exposure. We established oats-avenin-specific and -reactive intestinal T-cell lines from these three patients, as well as from two other patients who appeared to tolerate oats. The avenin-reactive T-cell lines recognized avenin peptides in the context of HLA-DQ2. These peptides have sequences rich in proline and glutamine residues closely resembling wheat gluten epitopes. Deamidation (glutamine→glutamic acid conversion) by tissue transglutaminase was involved in the avenin epitope formation. Conclusions We conclude that some celiac disease patients have avenin-reactive mucosal T-cells that can cause mucosal inflammation. Oat intolerance may be a reason for villous atrophy and inflammation in patients with celiac disease who are eating oats but otherwise are adhering to a strict gluten-free diet. Clinical follow-up of celiac disease patients eating oats is advisable.	Introduction Celiac disease is a chronic inflammatory condition caused by an inappropriate immune response of intestinal T-cells reactive to gluten proteins of wheat and similar prolamin proteins of related cereals [ 1 ]. The majority of the peptides recognized by intestinal T-cells are more immunogenic following deamidation by tissue transglutaminase (TG2). These peptides are invariably presented by HLA-DQ2 or -DQ8, the same HLA molecules that confer genetic predisposition to celiac disease [ 1 ]. Gluten-reactive intestinal T-cells can be isolated from virtually all patients with celiac disease but not from normal individuals. The disease goes into remission when harmful cereals are avoided. A gluten-free diet is thus the standard treatment of this disorder. Oats have traditionally been excluded from the gluten-free diet. Several feeding studies, however, have indicated that patients with celiac disease and dermatitis herpetiformis tolerate oats without signs of intestinal inflammation [ 2 – 9 ]. Of note, some of these studies have high patient-dropout rates that may have masked cases of oat intolerance. An in vitro study found no signs of T-cell activation in small intestinal biopsies of celiac disease patients challenged with avenin (the prolamin fraction of oats) [ 10 ], and avenins have been predicted to contain only a few glutamines that can be deamidated by TG2, presumably making avenins less immunogenic [ 11 , 12 ]. On this basis, oats have been allowed in the gluten-free diet in several countries [ 13 ]. It remains to be proven that all celiac disease patients tolerate oats following long-term exposure. A recent study of 39 Finnish patients randomized to eat a gluten-free diet with 50 g of oats daily or a standard gluten-free diet for 1 y reported more intestinal symptoms and more gut inflammation in the group of patients eating oats, although the mucosal integrity was not disturbed [ 14 ]. In an open challenge study of 19 adult celiac disease patients using pure oats, one patient developed villous atrophy [ 15 ]. This finding prompted us to investigate the phenomenon of oat intolerance further in a selected series of nine adult celiac disease patients, three of whom had clinical oat intolerance. The goal of the study was to characterize the intestinal T-cell response to oats avenin proteins in these patients in detail and to relate this to clinical symptoms and intestinal biopsy findings. Methods Participants We studied nine adults with celiac disease who had a history of exposure to pure oats. The oats were derived from a quality-controlled production line and were shown to be free from contamination of other cereals as described elsewhere [ 15 ]. The selection of the study participants was not random. Five of the patients (CD359, CD377, CD422, CD431, and CD482) participated in a clinical challenge study consisting of 19 adults with celiac disease who ate 50 g oats daily for 12 wk [ 15 ]. One of these patients (CD422) has symptoms and mucosal inflammation on oats consumption as described [ 15 ]. Patient CD431 has slight mucosal inflammation when eating oats but is clinically well. The three remaining individuals eat and tolerate oats. All these five patients agreed to undergo gastroduodenoscopy for research purposes. In addition, two other adults with celiac disease (CD446 and CD504) were recruited from our ordinary outpatient clinic. Patient CD446 eats and tolerates oats, whereas patient CD504 has anaphylactoid symptoms after intake of oats but has no mucosal inflammation. Finally, two patients (CD496 and CD507) were referred by a general practitioner and a referring hospital for investigation of complications arising when eating a gluten-free diet, here termed complicated celiac disease. The latter four patients came for gastroduodenoscopy for clinical reasons, and agreed to have extra biopsies taken for research purposes. We were unable to measure serological parameters in these last four patients because no serum samples were taken from them during their clinical course. The study was approved by the regional ethical committee. The participants gave their informed consent. Histopathological Assessment We took small intestinal biopsies from the horizontal part of the duodenum by gastroduodenoscopy using an Olympus (Tokyo, Japan) GIF-IT140 scope and scored them according to the modified Marsh criteria [ 16 ]. Intraepithelial lymphocytes were counted in hematoxilin-eosin-stained sections and enumerated per 100 enterocytes. Five areas per biopsy were counted, each encompassing 50–100 epithelial cells. Grain Antigens and Peptides Oat grains (Regal, Oslo, Norway) were ground and the flour was washed twice with water-saturated 1-butanol. The pellet was dissolved in 45% ethanol overnight and centrifuged. The avenin fraction was precipitated from the supernatant by adding two volumes of 1.5 M NaCl. The precipitate was dissolved either in 0.01 M acetic acid (pH 1.8) and digested with pepsin and subsequently trypsin (pH 7.8) or in 2 M urea /0.01 M NH 4 HCO 3 and digested with chymotrypsin. Gluten and gliadin (ethanol-soluble proteins of gluten) were isolated from household wheat flour and digested with chymotrypsin as described [ 17 ]. Avenin peptides were synthesized on a robotic system (Syro MultiSynTech, Bochum, Germany) using Fmoc/OtBu chemistry and 2-chlorotrityl resin (Senn Chemicals, Dielsdorf, Switzerland). The identity of the peptides was confirmed by electrospray mass spectrometry, and purity was analyzed by reverse-phase HPLC. Treatment of the peptides with guinea pig (Sigma; St. Louis, Missouri, United States) and human recombinant TG2 was performed in the presence of 1 and 2 mM CaCl 2 , respectively, at 37 °C for 2 h. T-Cell Assays The generation of T-cell lines, T-cell cloning, and T-cell proliferation assays were performed as described elsewhere [ 17 ]. Single biopsy specimens from the patients were challenged in vitro with either a pepsin-trypsin digest or a chymotrypsin digest of avenin. As control, single biopsy specimens were challenged with a chymotrypsin digest of gluten or gliadin. DR3+DQ2+ B lymphoblastoid cells (irradiated 80 Gy) were used as antigen-presenting cells. Positive T-cell responses were defined as a stimulatory index (SI) ([T + APC + antigen] divided by [T + APC]) above 3. Determination of HLA restriction of the T-cells was done by testing inhibition of T-cell proliferation by the monoclonal antibodies B8.11 (anti-DR), SPV-L3 (anti-DQ), 2.12.E11 (anti-DQ2), and W6/32 (anti-HLA class I). Purification of Avenin Fragments A pepsin-trypsin digest of avenin was separated by gel filtration (FPLC, column Superdex Peptide HR 10/30; Pharmacia Biotech, Uppsala, Sweden), and a fraction containing T-cell stimulatory fragments was further separated by reverse-phase HPLC (Äkta, Pharmacia Biotech; column Jupiter 5μ C18, 250 × 4.6 mm, Phenomenex, Torrance, California, United States) using an acetonitrile gradient from 5% to 40% with 1%/min and from 40% to 64% with 3%/min (flow rate 0.9 ml/min, containing 0.05% trifluoroacetic acid). Mass Spectrometry and Database Searching Electrospray ionization tandem mass spectrometry was performed on a quadrupole time-of-flight hybrid mass spectrometer (Micromass, Manchester, England). For spraying, needles were typically held at 900 V towards a skimmer cone (40 V). In collision-induced dissociation of selected peptide ions (collision gas argon; collision energy 25–35 eV), the generated characteristic b- and y-type fragment ions [ 18 ] were detected by the orthogonal TOF mass analyzer. All tandem mass spectrometry spectra were centroided and searched against in the NCBInr database via the Mascot Search Engine ( http://www.matrixscience.com ). Results Clinical and Histological Characteristics Nine adults with celiac disease who had a history of exposure to oats assessed to be free from contamination of other cereals were studied. In some cases they came for gastroduodenoscopy for clinical reasons, in other cases, they agreed to come for research reasons. The characteristics of the patients are given in Table 1 . This case series is thus not a consecutive series of ordinary patients with celiac disease. Three of these patients (CD422, CD496, and CD507) were known to exhibit clinical and histopathological signs of oat intolerance. Patient CD422 developed villous atrophy and dermatitis while eating oats, and details of this patient are described elsewhere [ 15 ]. Patient CD496 was a 53-y-old woman who was evaluated for complicated celiac disease. Celiac disease was diagnosed in 1987 after 1 y with diarrhea and weight loss; a biopsy showed a Marsh 3C lesion with an intraepithelial lymphocyte (IEL) count of 58/100 enterocytes (range 53–69) ( Figure 1 ). She responded well to a standard gluten-free diet. A control biopsy was not taken. In 2001, she started eating pure oats, but lost weight, going from 55 kg to 44 kg. While eating oats, a biopsy showed a Marsh 3A lesion with an IEL count of 54/100 enterocytes (range 43–62). The oats were discontinued, and she gradually recovered. Some months later, an intestinal biopsy demonstrated a Marsh 1 lesion with an IEL count of 46 (range 28–52). Clinically she is currently well. Patient CD507 was a 59-y-old woman who was also evaluated for complicated celiac disease. She probably had undiagnosed celiac disease since childhood and was diagnosed in 1990 after osteoporotic fractures. A biopsy showed total villous atrophy (Marsh 3C) with an IEL count of 50/100 enterocytes (range 44–54) ( Figure 1 ). She responded well to a standard gluten-free diet. In 1999, a follow-up biopsy showed complete normalization of her mucosa (Marsh 0) with an IEL count of 26/100 enterocytes (range 24–32). In 2000, the patient started eating oats and developed bloating, abdominal pain and iron deficiency. She lost 2 kg in weight. In 2002, while still eating oats, a biopsy showed a Marsh 3A lesion with an IEL count of 50/100 enterocytes (range 38–58). She discontinued eating oats and improved clinically. A new biopsy later in 2002 showed improvement, with a Marsh 1 lesion with an IEL count 32/100 enterocytes (range 24–46). Surprisingly, in late 2003 she was diagnosed with an adenocarcinoma in the small intestine, which was removed surgically. Figure 1 Histology of Intestinal Mucosa of Two of the Oat-Intolerant Patients Small intestinal biopsies were obtained at diagnosis, after an ordinary gluten-free diet (remission), after introduction of oats, and after withdrawal of oats (recovery). For patient CD496, a biopsy was not taken after she started with a gluten-free diet. Biopsies were scored according to the modified Marsh criteria. Hematoxilin-eosin staining was used, and IEL counts are given in the corners of the photomicrographs. The remission biopsy from patient CD507 was poorly oriented. We therefore melted and reoriented this biopsy (insert). Original magnification: 100×. Table 1 Characteristics of the Included Patients Avenin-Reactive T-Cell Lines Generated from Intestinal Biopsies Challenged with Avenin Responses to TG2-treated avenin were detected in the polyclonal T-cell lines derived from the avenin-challenged biopsies from all three patients who had clinical and histopathological signs of oat intolerance ( Table 1 ). Intestinal T-cell responses to TG2-treated avenin were also found in two of the other six patients. At least one avenin-reactive T-cell line from each patient was expanded. Inhibition experiments using anti-HLA class I and class II monoclonal antibodies demonstrated that these T-cell lines were all restricted to DQ2 (Figure 3; unpublished data), and with the exception of the T-cell line generated from patient CD482, they all gave an enhanced T-cell response to avenin treated with TG2 compared to avenin not treated with TG2 ( Table 2 ). T-cell lines derived from the biopsies challenged ex vivo with avenin gave higher responses to the TG2-treated avenin than to TG2-treated gluten in four of five patients (CD422, CD431, CD482, and CD496, but not CD507; Table 2 ). Notably, intestinal T-cells specific for TG2-treated wheat gluten and gliadin were identified in control biopsies challenged with gluten in all nine celiac patients (see Table 1 ). Table 2 T-Cell Responses to Avenin, Gluten, and Avenin Peptides in T-Cell Lines Established from Biopsies Stimulated with Avenin Antigen Both untreated and TG2-treated antigens were tested for recognition. The protein antigens were tested at 100 μg/ml, and the peptide antigens were tested at 10 μM. The responses are given as SIs ([T + APC + antigen] divided by [T + APC]), and positive responses are indicated by bold type Identification of a T-Cell Epitope in Avenin To identify the T-cell stimulatory peptides, we initially studied an avenin-specific T-cell line (TCL CD422.2.4) isolated from the oat-intolerant patient CD422. The T-cells weakly recognized one gel filtration fraction (#25) of a pepsin-trypsin digest of avenin. This fraction was further separated by reverse-phase HPLC, and retested for T-cell recognition ( Figure 2 A and 2 B). Stimulatory fractions were subjected to electrospray ionization tandem mass spectrometry ( Figure 2 C). For fractions 3 and 4, a single 22-mer peptide was identified differing only by an asparagine (fraction 3) and an aspartic acid residue (fraction 4). The two identified peptides from fraction 8 represent C-terminally elongated derivates of these 22-mers. Five peptides identified from fraction 9 correspond to N-terminally truncated and C-terminally elongated variants. Figure 2 Identification of an Epitope in Avenin Recognized by Intestinal T-Cells of Celiac Disease Patients (A) Reverse-phase HPLC of a pepsin-trypsin digest of avenin. Peptides in fraction 25, obtained from gel filtration, were eluted by an acetonitrile gradient (straight line), and 41 fractions were collected. Fractions recognized by T-cell lines in subsequent experiments are indicated by the numbers above the peaks. (B) T-cell recognition of fractions obtained by reverse-phase HPLC. All 41 fractions obtained in (A) were tested for recognition by the intestinal T-cell line 422.2.4 (derived from an oat-intolerant celiac disease patient). The fractions were incubated with DR3-DQ2 homozygous antigen-presenting cells overnight before the T-cell line was added. Specific T-cell responses were measured by 3 H-thymidine incorporation. Pepsin-trypsin-digested avenins, both TG2-treated and untreated, were used as control antigens. (C) Sequences of the peptides in the stimulatory fractions from reverse-phase HPLC identified by tandem mass spectrometry and overlapping synthetic peptides used for T-cell assays. For better comparison, the amino acid sequence of the avenin precursor protein JQ1047 (gi82331) is taken as a consensus sequence, and deviating residues are underlined. T-Cell Recognition of Synthetic Avenin Peptides Four avenin peptides (1488, 1489, 1490, and 1491; Figure 2 C) almost completely covering the sequences identified in the reverse-phase HPLC fractions 3, 4, 8, and 9 were synthesized and tested for T-cell recognition. Only peptide 1490 (SEQYQPYPEQ Q EPFVQQQQ) was recognized by the T-cell lines CD422.2.4, CD496.2.1, and CD431.2 (see Table 2 ; Figure 3 ). The recognition of this peptide by the T-cell lines CD422.2.4 and CD431.2 was dependent on TG2 treatment. T-cell line CD496.2.1 responded to the native peptide, but the response was augmented by treatment with TG2. We identified one deamidation site by tandem mass spectrometry (underlined in the above given sequence). This regioselectivity of deamidation conforms to the previously defined specificity of TG2 [ 11 , 19 ]. Several truncation variants of peptide 1490 were also synthesized. The shortest peptides tested were 12-mers; the T-cell line CD431.2 recognized peptide 1505 (YQPYPEQQEPFV) after TG2 treatment and the already deamidated peptide 1504 (YQPYPEQEEPFV) without TG2 treatment ( Figure 3 ). We predict the 9-mer core region binding to DQ2 as PYPEQEEPF, placing the glutamic acid resulting from the deamidation at the P6 position. This is similar to the DQ2-α-I gliadin epitope (PFPQPELPY), which also binds to DQ2 with a glutamic acid at the P6 position [ 20 , 21 ]. Recently, Vader et al. studied whether T-cells generated from celiac disease biopsies stimulated with wheat gluten would cross-react with predicted epitopes of barley, rye, and oats. From these studies they found two broadly reactive polyclonal T-cell lines that responded to peptides from barley hordeins, rye secalins, and the avenin-derived peptides Av-α9A, which is identical to a length variant of 1490 (QYQPYPEQQEPFVQ), and Av-α9B (QYQPYPEQQQPFVQ) [ 22 ]. We tested peptide Av-α9B against our T-cell lines and found that it was recognized by T-cell lines from the patients CD422 (line 2.4), CD496 (lines 2.1 and 2.3), and CD507 (line 2.3) after TG2 treatment ( Table 2 ; unpublished data). From the T-cell line CD496.2.3, we generated a T-cell clone that was specific for the peptide Av-α9B after TG2 treatment ( Figure 4 A). This clone responded also to TG2-treated avenin, but did not display cross-reactivity to TG2-treated gluten nor to the TG2-treated 1490 peptide ( Figure 4 B). The avenin-reactive T-cell line generated from the patient CD482 (CD482.2.1) did not recognize the 1490 peptide nor the Av-α9B peptide. Thus, there exist at least two distinct peptides of oats that can elicit mucosal T-cell responses in celiac disease patients with clinical intolerance to oats. Figure 3 HLA Restriction and Avenin Peptide Specificity of the Intestinal T-Cell Line 431.2 from Patient CD431 The avenin antigen (at 0.25 mg/ml) and peptides (at 10 μM), treated with TG2 when indicated, were incubated overnight with DR3-DQ2 homozygous antigen-presenting cells before T-cells were added. In the HLA restriction experiments, anti-DR or anti-DQ monoclonal antibodies were added 30 min prior to the T-cells. T-cell responses were measured by 3 H-thymidine incorporation and are represented as SIs. Figure 4 Reactivity of an HLA-DQ2-Restricted T-Cell Clone Derived from a T-Cell Line (CD496.2.3) Established by Avenin Stimulation of an Intestinal Biopsy of Patient CD496 T-cell responses were measured by 3 H-thymidine incorporation and are represented as SIs. (A) The clone specifically recognizes the avenin peptide Av-α9B after treatment with TG2. The peptides were tested at 10 μM. (B) The clone recognizes avenin but not gluten antigen after treatment with TG2. Location of Epitopes to a Proline- and Glutamine-Rich Region of Avenin The avenin epitopes we identified are localized to a region of avenin uniquely rich in proline and glutamine residues ( Figure 5 ). The presumed 9-mer core region of the avenin epitopes (PYPEQQEPF and PYPEQQQPF) contains three proline residues. The high number of proline residues and the localization of the epitopes to a region rich in proline and glutamine residues bear strong resemblance to features typical of DQ2-restricted T-cell epitopes of wheat gliadin [ 23 , 24 ]. Figure 5 Amino Acid Sequence of an Avenin (gi 82331, JQ1047) and an α-Gliadin (α2-Gliadin, AJ133612) The proline and glutamine residues are red and blue, respectively. In the avenin, the presumed 9-mer core region of the characterized T-cell epitope is underlined. In the α-gliadin, a 33-mer natural fragment containing six copies of three partly overlapping epitopes (DQ2-α-I, PFPQPQLPY; DQ2-α-II, PQPQLPYPQ; and DQ2-α-III, PYPQPQLPY) is underlined. Note the localization of all the epitopes to regions of the proteins rich in proline and glutamine residues and the high number of proline residues within the 9-mer core regions of the epitopes. Discussion A number of previous reports concluded that all celiac disease patients tolerate oats. These reports have formed the basis for approving oats in the gluten-free diet for the treatment of celiac disease. The findings reported here demonstrate that oat intolerance exists in some celiac disease patients, and the study provides a molecular explanation for this intolerance. Oats are less related to wheat than are barley and rye. In oats, the prolamines represent much less of the total seed proteins than in the other cereals [ 25 ]. In addition, avenins contain about half the amount of proline residues (10%) as the prolamins of wheat (gliadins and glutenins), barley (hordeins), and rye (secalines). On this basis, it is intriguing that the identified avenin epitopes are located in the regions of avenins with the highest content of proline residues, regions also rich in glutamine. This is analogous to the localization of the T-cell epitopes in α- and γ-gliadins [ 23 ]. The immunogenicity of gliadin peptides is influenced both by the glutamine residues, which become specifically deamidated by TG2, and by the proline residues, which protect the peptides from proteolysis in the gastrointestinal tract, determine the specificity of TG2, and are crucial for the selective binding to HLA-DQ2 [ 1 ]. This study shows that the same features apply to T-cell epitopes of avenin. In humans it is impossible to directly demonstrate that T-cells induce disease. In celiac disease this relates equally to T-cells reactive to gluten and to T-cells reactive to avenin. The fact that avenin-reactive intestinal T-cells, like gluten-reactive T-cells from celiac disease patients, are uniquely restricted by HLA-DQ2 and are activated by TG2-treated peptides speaks strongly in favor of their involvement in the disease pathogenesis. The finding of avenin-specific intestinal T-cells also in individuals with celiac disease that are clinically tolerant to oats does not, as we see it, contradict this assumption. Some patients with celiac disease stay in remission for extended time periods during gluten challenge even if it is likely that they have gluten-reactive T-cells in their intestinal mucosa. Since avenin is less immunogenic than wheat gluten, one would expect an extended time for relapse to be at least as common during oats consumption. It is highly unlikely that the intolerance and the mucosal inflammation observed in our patients could be explained by contamination of the oat flour by wheat, barley, or rye proteins. All the oats consumed were produced in a quality-assessed production line. Our data indicate that avenin can drive mucosal inflammation in that the incubation of the intestinal biopsies with avenin enriches for activated, avenin-reactive T-cells. A substantial proportion of the avenin-reactive T-cells appear to be specific to avenin. The T-cell clone we established from an avenin-challenged biopsy was reactive to avenin but did not cross-react to wheat gluten, and the T-cell lines from biopsies challenged with avenin responded more strongly to avenin than to gluten in four of five participants. Cross-reactivity at the T-cell clonal level has been demonstrated between wheat gluten, hordein, and secalin antigens [ 22 , 26 ] and likely also exists between gluten and avenin [ 22 ]. Even if some of the avenin-reactive T-cells were originally primed to gluten and responded to avenin because of cross-reactivity, they would still participate in an avenin-driven immune response. T-cell reponses to the avenin epitopes described in this paper have been found in T-cell lines derived from intestinal biopsies of patients with celiac disease that were stimulated with gliadin [ 22 ]. It is unknown whether any of the patients from whom these T-cells were isolated had clinical symptoms or mucosal inflammation related to oats ingestion. Thus, to our knowledge, the current study is the first to demonstrate a mechanistic link between clinical symptoms of oat intolerance, mucosal inflammation, and avenin-reactive T-cells. Oat intolerance can cause complications in the large group of celiac disease patients who are now regularly consuming oats. At this stage we do not know how frequently such complications may occur. Presumably such complications will not be very common, but only extended clinical follow-up of oats-consuming celiac disease patients will establish the frequency. Monitoring of T-cell responses to avenin epitopes may potentially identify individuals who are at risk of developing oat intolerance. Based on our data, such monitoring will also identify some individuals who are clinically tolerant to oats and who have minimal or no mucosal pathology after a limited oats challenge. Possibly some of these patients may have latent oat intolerance that will develop into overt disease after prolonged exposure, but this remains speculative. Our observations demonstrate that even if oats seem to be well tolerated by many celiac disease patients, there are patients who have an intestinal T-cell response to oats. Until the prevalence of oat intolerance in celiac disease patients is established, clinical follow-up of celiac disease patients eating oats is advisable. Clinicians should be aware that oat intolerance may be a reason for villous atrophy and inflammation in patients with celiac disease who are eating oats but otherwise are adhering to a strict gluten-free diet. Patient Summary Background Celiac disease is a digestive disease that damages part of the gut (the small intestine) and interferes with absorption of nutrients from food. Patients with celiac disease do not tolerate a protein called gluten, which is found in wheat, rye, and barley. When people with celiac disease eat foods containing gluten, their immune system responds by damaging the small intestine. The disease is quite serious in some patients, but eating a strictly gluten-free diet can eliminate all of the symptoms. Unfortunately, wheat, barley, and rye products like flour are found in many common foods, and patients have to avoid them for the rest of their lives. Previous studies suggested that oats were safe for patients with celiac disease, and as a result, they often form part of a gluten-free diet. What Did the Researchers Find? Contrary to other studies, this one demonstrates that oats intolerance does exist in some patients with celiac disease. These patients have an immune reaction to oats that is similar to the reaction most celiac disease patients have to wheat, barley, and rye. What Does This Mean for Patients? It appears that oats are not safe for all patients with celiac disease. Patients who eat oats as part of a gluten-free diet should discuss their diet and any symptoms with their doctors; doctors should keep in mind that patients might develop symptoms when they eat oats. What Are the Problems with the Study? The researchers studied only a small number of patients, and this study cannot tell us how common oats intolerance is among celiac disease patients. Where Can I Find More Information? US National Institutes of Diabetes, Digestive, and Kidney Disorders: http://digestive.niddk.nih.gov/ddiseases/pubs/celiac/ Celiac Disease Foundation: http://www.celiac.org/ The Gluten Intolerance Group: http://www.gluten.net/ The Celiac Disease Foundation: http://www.celiac.com/	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC523824.xml
515372	Mitochondrial Genes Cause Nuclear Mischief	null	While the nucleus of a cell may be its command headquarters, mitochondria are equally vital—they are the power plants of the cell, and without them all cellular activity would quickly and irrevocably come to a halt. Testifying to their origins as once free-living bacteria, mitochondria have their own DNA, comprising 37 genes in humans on a single circular chromosome. Whether they invaded their ancestral hosts as parasites or were captured as subcellular collaborators, they have long since left their independent ways behind. Their meager complement of genes is far fewer than is needed to produce these complex organelles; it is clear from analyzing the nuclear genome that most of the mitochondria's presumed ancestral genes have been taken into the cell's nucleus, where they are under the strict control of their host. The transplanted mitochondrial genes have been faithfully doing their job under new management since they were first appropriated, probably hundreds of millions of years ago. However, not all of their DNA descendants have continued to make themselves so useful. For, in addition to many of the mitochondria's original genes, the human genome houses over 200 mitochondrial genetic fragments, useless pieces of code whose only remaining function is to be replicated generation after generation. Detritus from other sources is even more common within the genome, and most of it seems to be harmless. But in this issue, Ricchetti and colleagues show that mitochondrial fragments may not be quite so benign. They have continued to invade the human genome, even into the present day, and a large proportion of them take up residence within nuclear genes, possibly disrupting them and causing human diseases. Scanning the entire human genome, Ricchetti and colleagues found a total of 211 nuclear sequences of mitochondrial origin (NUMTs). Of these, they selected 42, which appeared to be the most recent integrations, for detailed study. Only 14 of them were also found in DNA from our closest relatives, chimpanzees, indicating that the rest arose after the human–chimp split approximately 5 million years ago. While 35 of the 42 were found in all humans tested, the rest were not, suggesting a still more recent origin for these among human populations. The authors also made two surprising discoveries about the location of these human-specific NUMTs. They were not evenly distributed across the entire genome; instead, for reasons that are unclear, there were a disproportionate number of them on two chromosomes—the Y chromosome, present only in males, and number 18. Furthermore, NUMTs were not randomly scattered among all the DNA of the chromosomes. Rather, they were much less likely to be found in non-coding “junk” DNA and much more likely to have inserted themselves within highly active genes. This phenomenon is likely to be related to the mechanism by which a NUMT enters the chromosome—it relies on the machinery that repairs breaks in the DNA, and these breaks are more common in genes that are frequently transcribed. Such insertions can cause disease, as shown by the recent discovery of a hemophilia patient with a NUMT interrupting his clotting factor gene. Much remains to be learned about the functional and temporal dynamics of NUMT insertions, but their potential for harm suggests that many NUMTS, unlike much of the rest of the flotsam that litters our genome, may be selected against quickly. Combined with their differential distribution among human ethnic groups, this may make them valuable markers for tracking both long- and short-term trends in human evolution and migration.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC515372.xml
549586	Individual Level Injection History: A Lack of Association with HIV Incidence in Rural Zimbabwe	Background It has recently been argued that unsafe medical injections are a major transmission route of HIV infection in the generalised epidemics of sub-Saharan Africa. Methods and Findings We have analysed the pattern of injections in relation to HIV incidence in a population cohort in Manicaland in a rural area of Zimbabwe. In Poisson regression models, injections were not found to be associated with HIV in males (rate ratio = 0.33; 95% confidence interval: 0.07 to 1.46) or females (rate ratio = 1.04; 95% confidence interval: 0.59 to 1.85). Conclusion It is important that unsafe medical injections can be confidently excluded as a major source of HIV infection. In rural Zimbabwe the evidence is that they can.	Introduction The widely held belief that heterosexual transmission is the driving force behind sub-Saharan Africa's (SSA's) HIV epidemic [ 1 ] has recently been questioned [ 2 ]. According to Gisselquist and colleagues, investigators have overlooked the importance, and indeed suppressed analysis, of unsafe medical injections as a route of transmission for HIV. Hitherto, assessments of this hypothesis have largely relied on ecological analyses—relating population-level data on unsafe injections to the distribution of HIV prevalence [ 3 , 4 ]. The absence of investigation into the role of unsafe injections, based on the assumed predominance of sexual transmission, has rightly been criticised. However, this criticism trivialises the difficulty of collecting and analysing relevant field data. Presently, the only published data on the possible contribution of injections to HIV transmission in SSA come from rural Uganda, where Kiwanuka et al. demonstrated no association of injections with HIV incidence [ 5 ]. Data from other SSA countries with generalised epidemics where spread has varied in scale and pattern are required to inform this debate. In this paper, we analyse data from a population cohort in Manicaland in rural Zimbabwe. We describe the determinants of injections in adults and then test the association between injections and incidence of HIV infection. Methods Data were analysed from the baseline (1999/2000) and follow-up (2002/2003) rounds of a cohort of adults in the Manicaland HIV/STD Prevention Study. Eligible men and women aged 15 to 54 were recruited based on an initial household survey [ 6 ]. In response to the awakening controversy, a question exploring exposure to injection was added two-thirds of the way through follow-up. Thus, data were available from individuals from four of the 12 study sites. In these sites, 505 males and 1,342 females were interviewed, representing a follow-up of 69.7% of individuals interviewed at baseline. The subset of individuals who were HIV-negative at baseline ( n = 1,606; 83.6%) was used for all analyses except for the examination of rates of injections stratified by HIV status at baseline. At follow-up, participants were asked whether they had received an injection or had been pricked by a needle since the baseline interview. A range of health and socio-demographic data were also collected, including self-reported history of sexually transmitted disease (STD) symptoms. Reports on STD symptoms were from the 1-y period before the follow-up interview, and thus did not correspond to the entire 3-y follow-up period. HIV serological testing was performed on dried blood spots using a highly sensitive and specific antibody dipstick assay [ 7 ]. Ethical approval for this study was granted by the Medical Research Council of Zimbabwe (MRCZ/A/681) and the Applied and Qualitative Research Ethics Committee, Oxford University, United Kingdom (N97.039). Written informed consent was sought from study participants. The objective of this analysis was to test the plausibility that injections are an important risk factor for HIV incidence. Having received an injection was modelled as a proximate cause with demographic variables, sexual behaviour, and STDs acting as potential confounders. Determinants of injections were analysed with univariable and multivariable Poisson regression models of incidence rate ratios (RRs). Attributes were retained in age-adjusted multivariable models if the stratum-specific RR differed from one and had a Wald-test p -value ≤ 0.1. Using the same strategy, models were then fitted with HIV as the outcome variable. Results Overall, 744 out of 1,847 individuals (40.3%) reported having received an injection or needle prick during the 3-y follow-up period. Females reported more injections than males (RR = 1.93). Rates were not significantly higher for individuals who were HIV positive at baseline (RR = 1.07, p = 0.81 for males; RR = 1.13, p = 0.28 for females) ( Table 1 ). Table 1 Univariable and Multivariable Poisson Regression Models of Incidence of Injections and Needle Pricks—Presented Separately for Males and Females Reported injections and person years may not add up across different attributes because of missing data a Adjusted for age and attributes significant at p ≤ 0.1 (Wald test) b Exposure between baseline and follow-up (approximately 3 y) c Exposure in the 1 y prior to follow-up d Not significant but shown to demonstrate the direction of effect CI, confidence interval; PYAR, person years at risk Being widowed, separated, or divorced was the only attribute associated with increased rates of injections for males ( Table 1 ). For females, STD symptoms and childbearing/pregnancy were significant in adjusted models ( Table 1 ). There were 67 HIV seroconversions (48 females and 19 males); 13 (19%) of those seroconverting reported not having had sex in the inter-survey period, and 40 (60%) reported not having had an injection during the period ( Table 2 ). Table 2 Univariable and Multivariable Poisson Regression Models of HIV Incidence—Presented Separately for Males and Females Seroconversions and person years may not add up across different attributes because of missing data a Adjusted for age and attributes significant at p ≤ 0.1 (Wald test) b Exposure between baseline and follow-up (approximately 3 y) c Not significant but shown to demonstrate the direction of effect d Exposure in the 1 y prior to follow-up CI, confidence interval; PYAR, person years at risk There was no significant association between injections and HIV incidence among either males or females—in either unadjusted or adjusted models. For males, HIV seroconversion rates were elevated amongst 25- to 44-y-olds, sexually active individuals, and those who had suffered STD symptoms, though none of these attributes reached levels of statistical significance ( Table 2 ). For females, having multiple sexual partners, having STD symptoms, and being widowed/separated/divorced were associated with increased HIV incidence. Childbearing/pregnancy, which was strongly associated with injections (see Table 1 ), had no association with HIV incidence ( Table 2 ). Discussion These data, from a population cohort in rural Zimbabwe, suggest that—at the population level—injections are not a major route of HIV transmission. There was a very slight, non-significant association between injections and HIV amongst females. Could this association achieve significance with a greater sample size or more events? Given the strong association between STD symptoms and both injections and HIV incidence, this is possible, but would likely be a result of residual confounding. In other words, both HIV and injections have a common association with STDs. It has been argued that the association between sexual activity and HIV is confounding between STDs and pregnancy and injections and that it is the injections that are causal. Our analysis does indeed find these associations—but finds STD symptoms the strongest predictor of new HIV infections. Our measure of injection risk is unambiguous but lacks many dimensions relating to unsafe injections. Presently, we have collected data about the receipt of injections and other needle pricks. Thus, the exposures in these analyses are not restricted to injections received from the health-care sector—the source that Gisselquist et al. originally hypothesized as a major route of transmission [ 2 ]. Also, these data reflect only whether an individual had an injection or not—rather than the number of injections received. World Health Organization estimates suggest that the number of injections people receive is not evenly distributed in SSA populations [ 8 ]. Thirteen of 67 individuals seroconverting in this study reported no sexual partners in the inter-survey period. Only four of these 13 reported never having had sex. This leads us to suspect that incorrect categorisation of HIV status at baseline in addition to misreporting of sexual behaviour may explain some of these infections: it is possible that a proportion of the nine individuals who reported having had sex in their lifetime but not in the inter-survey period had been recently infected but had not yet seroconverted at the time of the baseline testing [ 9 ]. In this scenario, their exposure would have occurred prior to baseline, rather than in the follow-up period. Recall biases may also play a role, given the relatively long follow-up of 3 y [ 10 ]. Non-regular partners, especially those from the beginning of the recall period, may have been under-reported. Eliciting accurate reporting of sexual activity is notoriously difficult in Africa and elsewhere, though the use of informal confidential voting interviews has decreased social desirability biases in this cohort [ 11 ]. Nonetheless, the cases ( n = 4) where individuals seroconverted who had reported never having had sex may still be a product of social desirability reporting bias. Clearly, incidence data offer the most explanatory power in elucidating the determinants of the HIV epidemic, but these anomalous cases also highlight the difficulties of collecting time-varying sexual behaviour and serostatus information. Interestingly, in light of expected HIV-associated disease and care, individuals who were HIV positive at baseline did not have higher rates of injections than individuals who were HIV negative. Injections were found to be highly associated with childbirth/pregnancy. HIV-positive women—and especially those at advanced stages of infection—are known to experience reduced fertility [ 12 ]. Therefore, a reduction in use of maternal health services may partially explain why injections were not more common in the HIV-positive population. A more discriminating measure of exposure, including the reason for injection, could help to explain this observation. Had injections proven to be a risk factor for HIV incidence, further investigations would have been needed to determine the source and types of risky needle pricks. However, no such association was found. Unsafe injections are unacceptable, but this evidence suggests that they do not play a major role in the transmission of HIV in rural Zimbabwe. Patient Summary Background There is a lot of controversy over whether the spread of HIV in sub-Saharan Africa is predominantly caused by unsafe sexual practices, or whether unsafe medical injections given by health professionals might also have a prominent part to play. A recent paper suggested that unsafe medical injections were important. What Did the Authors Do? In an ongoing survey in rural Zimbabwe between 1999/2000 and 2002/3 that was trying to assess why some individuals get infected with HIV, the authors asked 505 men and 1,342 women a number of questions. They asked them about their sexual history, whether they had children, and whether they received injections. They tested the adults for HIV at the beginning and end of the study period. What Did They Find? 744 people had had a medical injection and 67 people acquired HIV. There was no evidence overall that injections were linked with an increase in HIV infection. The strongest link with HIV infection was with symptoms of sexually transmitted diseases (in other words, people with these symptoms were more likely to acquire HIV infection). What Do These Findings Mean? These findings suggest that although it is still possible for an individual to get HIV through unsafe medical injections, overall in this population in Zimbabwe, unsafe injections are not an important cause of HIV infection. Hence policymakers should concentrate more on trying to prevent infection from unsafe sex. Where Can I Get More Information? Information on safe sex: http://www.thebody.com/safesex.html World Health Organization Web page on reducing the risk of HIV infection in drug users who inject drugs intravenously: http://www.who.int/hiv/topics/harm/reduction/en/ Fact sheet from the Joint United Nations Programme on HIV/AIDS on HIV/AIDS in Zimbabwe: http://www.unaids.org/html/pub/publications/fact-sheets01/Zimbabwe_en_pdf.htm	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549586.xml
544897	Variations and voids: the regulation of human cloning around the world	Background No two countries have adopted identical regulatory measures on cloning. Understanding the complexity of these regulatory variations is essential. It highlights the challenges associated with the regulation of a controversial and rapidly evolving area of science and sheds light on a regulatory framework that can accommodate this reality. Methods Using the most reliable information available, we have performed a survey of the regulatory position of thirty countries around the world regarding the creation and use of cloned embryos (see Table 1 ). We have relied on original and translated legislation, as well as published sources and personal communications. We have examined the regulation of both reproductive cloning (RC) and non-reproductive cloning (NRC). Results While most of the countries studied have enacted national legislation, the absence of legislation in seven of these countries should not be equated with the absence of regulation. Senator Morin was not correct in stating that the majority of recent legislation bans both RC and NRC. Recent regulatory moves are united only with regard to the banning of RC. While NRC is not permitted in seventeen of the countries examined, it could be permitted in up to thirteen countries. Conclusions There is little consensus on the various approaches to cloning laws and policies, and the regulatory position in many countries remains uncertain.	Background "The immense majority of countries who have passed legislation recently do ban both reproductive and therapeutic cloning" (Senator Morin, The Standing Senate Committee On Social Affairs, Science And Technology, Ottawa, Canada, Wednesday 18 February, 2004) . In February 1997 an article was published in Nature announcing the birth of what was to become the most famous sheep in history [ 1 ]. That sheep, known as Dolly, was the product of asexual reproduction. As the world's media unhesitatingly announced, she was a clone. The prospect of a human clone led to immediate calls for regulatory controls on the technology. There were, however, divisions, particularly when it became apparent that the potential uses of the technique were not limited to reproduction. Other potential uses came one step closer when, in the following year, it was announced that embryonic stem cells had been successfully extracted from non-cloned human embryos [ 2 ]. Now, a year after the death of Dolly [ 3 ], it is appropriate to review the current regulatory position on the creation and use of cloned embryos around the world [ 4 , 5 ] – particularly considering the public debate that has surrounded the recent cloning experiments in Korea, the granting of the first "research cloning" license in the UK, and the past and impending UN cloning debates. This paper is concerned with the creation of functional embryos, whether by nuclear transfer or embryo splitting. As this suggests, we will use 'embryo' to refer to any human entity considered theoretically capable of implantation and development in the womb. Many regulatory positions distinguish between the creation of a cloned embryo for reproductive purposes and for other purposes. For our purposes, the former will be called reproductive cloning ( RC for short ) and the latter non-reproductive cloning ( NRC for short ). This paper will examine the regulatory position of the thirty countries for which we have been able to obtain reliable information (see Table 1 ). Where possible, we have relied on copies of the original legislation or of English translations of that legislation. In some situations we have also found it necessary to rely on other published sources [ 6 ] and personal communications. Table 1 Summary of regulation* Country National legislation and effect, or approach in the absence of national legislation Australia Prohibition of Human Cloning Act 2002: Prohibits both RC and NRC Austria Act No.275 of 1st July 1992: Implicitly prohibits both RC and NRC Belgium Law of 11th May 2003: Prohibits RC but permits NRC Canada Assisted Human Reproduction Act 2004: Prohibits both RC and NRC China None Ministerial regulations prohibit RC and allow NRC Denmark Law No.460 of 1997: Prohibits both RC and NRC Finland Medical Research Act No.488 of 1999: Prohibits RC, but NRC might not be included within this prohibition France Law of July 2004: Prohibits both RC and NRC Germany Embryo Protection Act 1990: Prohibits both RC and NRC Greece Law 3089 of 23rd December 2002: Prohibits RC, but does not cover NRC Iceland Law No. 55 of 29th of May 1996: Prohibits both RC and NRC India None Guidelines reject RC but might allow NRC Ireland None Constitutional provision might prohibit both NRC and RC Israel Prohibition of Genetic Intervention (Human Cloning and Genetic Manipulation of Reproductive Cells) Law 1999: Imposes a moratorium on RC, silent on NRC Italy Law of 2003: Prohibits both RC and NRC Korea Bioethics Law 2003: Prohibits RC, silent on NRC Luxembourg None Mexico General Health Law of 7 May 1997: Prohibits both RC and NRC Netherlands Embryo Act 2002: Prohibits RC and imposes a moratorium on NRC New Zealand Medicines (Restricted Biotechnical Procedures) Amendment Act 2002: Prohibits RC, silent on NRC Norway Law No. 100 of 5 December 2003: Prohibits both RC and NRC Peru General Law No. 26842 of 9 July 1997: Prohibits both RC and NRC Portugal None Ratified Convention (discussed below) Russia Law on the Temporary Prohibition of Human Cloning 2002: Imposes a Moratorium on RC Spain Law 35 of November 1988: Prohibits both RC and NRC Sweden Law No. 115 of 14th March 1991: Implicitly prohibits both RC and NRC (with possible gaps) Switzerland Federal Law of 18 December 1998: Prohibits both RC and NRC Thailand None UK Human Fertilisation and Embryology Act 1990: Permits NRC under licence Human Reproductive Cloning Act 2001: Prohibits RC United States None Some states have legislation prohibiting RC or both RC and NRC * The International Digest of Health Legislation provides translations of the legislation of Austria (1993, vol 44); Denmark (1997, vol 48); Israel (2000, vol 51), Norway (2003, vol 54), Peru (1998, vol 49), and Switzerland (1999, vol 50, and 2003, vol 54). The Bulletin of Medical Ethics provides translations of the legislation of Finland (Feb 2000, p7) and Germany (Dec 1990, p9). In addition, we have used English translations of the legislation of Greece , the Netherlands , and Sweden (Ministry of Health and Social Affairs, Swedish Act concerning Use of Gene Technology on Human Beings and Experiments with Fertilised Ova , 1991). Understanding the complexity of this "regulatory patch work" is essential [ 7 ]. It provides a sense of the vast differences between nations, the issues on which there are different views, and the existing regulatory uncertainties. In addition, it highlights both the challenges associated with the regulation of a controversial and rapidly evolving area of science and the need for a regulatory framework that can accommodate this reality. Finally, it demonstrates that policy makers cannot rely on the existence of a single regulatory trend to inform policy development. Only on the banning of RC (reproductive cloning) do the world's legislatures and policy-makers display anything approximating a single mind. Senator Morin, who is a member of Canadian Senate, was not right to claim that a majority of recent legislation bans both RC and NRC (non-reproductive cloning). Even looking beyond recent legislation, only a narrow majority of the thirty countries studied actually prohibit NRC. What is more, a large minority of countries have yet to enact national legislation. This paper is divided into sections. The next, section II, will examine the degree of regulatory variation in the countries studied. Section III will ask whether the existence of legislation answers all regulatory questions. Using examples drawn from those countries with legislation, we seek to show how broad interpretative strategies are sometimes required to avoid unintended lacunae. Section IV uses examples to demonstrate the evolving nature of regulatory positions and increasing reliance on legislation. Section V examines the impact of international initiatives in European (ie the European Convention on Human Rights and Biomedicine) and the United Nations. Section VI will explain why the regulatory outcome usuallysays little about the ethical approach adopted by a particular jurisdiction. Section VII is the conclusion. Variation between countries As many commentators have noted, there is great variation in regulatory approaches even within countries that have decided to create relevant laws and policies [ 4 , 5 , 7 , 8 ]. No two countries have adopted identical regulatory measures on cloning, though the effect of those adopted in some countries is very similar. There is only one area of regulatory agreement – no jurisdiction has, yet, adopted legislation or guidelines permitting RC. As a result, there are essentially only two regulatory approaches to RC: prohibition or regulatory silence. Regulatory silence usually means that RC is technically legal in the jurisdiction in question, though if it were attempted, there would likely be a rapid regulatory response. The majority of the countries studied have now enacted national legislation (see Table 1 ). Only seven have yet to do so. The absence of national legislation in these seven countries should not, however, be taken to amount to an absence of regulation or, in the case of the US, an absence of state legislation. Legislation is just one of many possible regulatory responses. Ireland provides an illustrative example. The Eighth Amendment to the Irish Constitution (which forms Article 40.3.3) states that, "The State acknowledges the right to life of the unborn and, with due regard to the equal right to life of the mother, guarantees in its laws to respect, and, as far as practicable, by its laws to defend and vindicate that right." While this provision does not mention cloning, it has been taken to protect in vitro embryos and thereby prohibit NRC. In addition, doctors must comply with the guidance of the Medical Council, as this body has the power to remove their licence to practise in Ireland. The Medical Council's guidelines declare that " [t]he creation of new forms of life for experimental purposes or the deliberate and intentional destruction of human life already formed is professional misconduct" [ 9 ]. Also, it limits the manipulation of sperm or eggs to the "improvement of health" and adds that "if the intention is...the creation of embryos for experimental purposes, it would be professional misconduct" [ 9 ]. Thus, the absence of legislation in Ireland does not render all things permissible. Variation and non-reproductive cloning (NRC) NRC represents the source of considerable regulatory variation. While NRC is not permitted in seventeen of the countries studied, it could be permitted in up to thirteen other countries. Regulatory uncertainties make it impossible to be sure in some of these counties. Also, some countries (such as the US) have many jurisdictions, each capable of adopting a different regulatory position. Given the superficial similarity of many of these countries and jurisdictions, it is hard to explain such stark variation on cultural differences alone [ 8 , 10 ]. Only Belgium and the UK have deliberately enacted or extended legislation for the purpose of permitting the creation of cloned embryos for research [ 6 , 11 ]. The UK licensing authority has, in fact, granted its first licence to conduct NRC [ 12 ]. Similarly permissive approaches, albeit non-legislative, have been adopted by China (which issued Ministerial Regulations in August 2003 to allow cloning research for therapeutic purposes) [ 6 , 13 ] and Korea (where the government is in the process of approving limited research on limited somatic nuclear transfer research) [ 6 ]. In contrast, Finland, Greece, Israel, Russia and Sweden appear to allow NRC only because their legislation has potential gaps [ 14 ]. The Greece legislation is the most striking, because it is the most recently enacted. The Greek Law 3089/2002 explicitly prohibits 'human reproduction' by any cloning method, but no mention is made of NRC. This must have been deliberate, because the provision allowing embryo research does so by allowing research (with consent) on "fertilized ova" that are surplus following assisted reproductive treatment. Indeed, the legislation's Explanatory Memorandum declares that "only reproductive cloning is prohibited. It could be thus construed that therapeutic cloning is permitted....This position has also been supported in the Report of the National Bioethics Committee regarding the use of stem cells in biomedical research and clinical practice (21.10.2001), submitted to the Prime Minister on 11.1.2002." This is nonetheless controversial in Greece, which has ratified both the European Convention on Human Rights and Biomedicine, and its Additional Protocol on cloning (discussed below). The US, India, and Portugal are anomalous. In the US, what little national legislation there is only concerns the use of federal funds [ 4 ] and some States (such as California and New Jersey) have even adopted permissive legislation [ 15 ]. The Indian Council of Medical Research has declared that "research on cloning with intent to produce an identical human being, as of today, is prohibited", but has not declared NRC to be so prohibited [ 16 ]. However, an Indian Government policy document "opens the door to therapeutic cloning considered on a case-by-case basis by the National Bioethics Committee" [ 6 ]. Portugal has no national legislation, but has ratified the European Convention and its Additional Protocol (see below). Legislative gaps and uncertainties There are, of course, many nations that have long standing laws that are relevant to cloning technologies. In many of these nations, however, the laws were designed prior to Dolly and the recent advances in stem cell research. As such, how these laws might apply to cloning is sometimes unclear. Also, these laws are not a result of a public and political dialogue about the complex scientific and ethical issues that are associated with cloning and stem cell technologies. [ 10 ] In addition, there are a number of countries where recent legislative intervention has failed to answer all legal questions relating to human cloning. The legislation of some countries clearly encompasses somatic cell nuclear transfer (SCNT). The Spanish Law 35 of November 1998 is an example of a pre-Dolly legislation of this type. This Act not only renders it an offence to create identical human beings where it is aimed at race selection, it also renders it an offence to create "human beings by cloning in any of the variants or any other procedure capable of originating several identical human beings." The Canadian Assisted Human Reproduction Act 2004 is an example of post-Dolly legislation of this type. Under this Act, the creation and implantation of a "human clone" are prohibited. "Human clone" is defined under s. 3 to mean "an embryo that, as a result of the manipulation of human reproductive material or an in vitro embryo, contains a diploid set of chromosomes obtained from a single – living or deceased – human being, foetus or embryo". This clearly captures SCNT. In a number of contrasting countries, SCNT is only captured by a broad, non-literal interpretation of the relevant provisions. The Swedish Law No. 115 of 14 March 1991 is an example of pre-Dolly legislation of this type. This Act only regulates experiments performed on "fertilised ova" or gametes "before fertilisation". The Finnish Medical Research Act 1999 is a rare example of post-Dolly legislation of this type. This Act has many provisions with respect to research on embryos (including a prohibition on the creation of embryos for research) and it prohibits all research conducted with the aim of cloning a human being. However, s. 2 of the Act defines an embryo as "a living group of cells resulting from fertilisation not implanted in a woman's body". Thus, the Dolly technique only appears to be covered insofar as its use involves "research with the aim of cloning human beings" (Also, s.1 of the Constitution secures the inviolability of human dignity, but there is no authoritative interpretation on whether (and how) this provision could apply to cloning). If this aim is only correctly attributed to RC, then NRC might not be covered at all. The dangers of non-literal interpretation of pre-Dolly provisions should not be exaggerated. Although there are a number of countries that have such legislation (notably, Austria and Germany and, until very recently, France) [ 5 ], in reality the courts are likely to adopt a broad, purposive approach to interpretation. A very broad approach was, for example, taken when the domestic courts addressed the UK's Human Fertilisation and Embryology Act 1990 [ 17 ]. The Act explicitly prohibited only one form of cloning (the creation of a clone by replacing the nucleus of an embryo) leaving the licensing authority to regulate activities such as the creation, storage, and use of in vitro embryos. More precisely, the Act imposes a licensing requirement on the creation of an in vitro embryo (ss.3(1)(a) and 1(2)); storage or use of in vitro embryos (ss.3(1)(b) and 1(2)); storage of gametes (s.4(1)(a)); and use of gametes, unless 'services are provided for the woman and man together' (s.4(1)(b)). Yet, under s. 1(1) of the Act, "embryo" is defined as "a live human embryo where fertilisation is complete", including "an egg in the process of fertilisation". This raised the question of whether SCNT fell outside the Act altogether. Nonetheless, the House of Lords recently held that SCNT produces a functional embryo that falls within the ambit of this Act (in effect, holding that the Act's definition of embryo is non-exhaustive and restricted in purpose) [ 18 ]. Evolving nature of the laws Not only is there a great deal of variation between nations and much uncertainty as to the scope of existing laws, many of the existing laws and policies are in a state of flux. Indeed, some countries have built in review provisions. The Dutch legislation, the Embryo Act 2002, presents an example. This Act prohibits procedures undertaken for the purpose of creating genetically identical human individuals and prohibits the creation of embryos for research. Yet, s. 33 of the Act allows for the future repeal of the prohibition on the creation of embryos for research. Likewise, the recently enacted Canadian legislation states that a Parliamentary review of the law is required within three years of proclamation. Some countries, such as Israel, New Zealand and Russia, have even adopted time-limited legislation. The Israel legislation of 1999, for example, states that, for a period of 5 years , no intervention will be carried out on human cells for the purpose of human cloning or to bring about the creation of a person by the use of reproductive cells that have undergone permanent intentional genetic modification [ 19 ]. What is more, the Act states that the Minister of Health may (upon satisfaction of a number of conditions) permit the creation of a human being through the use of genetically modified cells. Non-legislative bans are often time-limited or chosen because of the ease with which they can be reconsidered. Other countries are in the process of considering a revision to their existing law. Ireland has set up a Commission on Assisted Human Reproduction in 2000 to explore this topic [ 20 ] and the Irish government has officially stated its opposition to cloning [ 21 ]. There are voices calling for revision of the Germany legislation [ 22 ]. The recently passed Italian legislation might have to be reconsidered because a referendum on a disputed law can be forced if 500,000 signatures are obtained and it has been reported that over a million people have signed a petition calling for a referendum [ 23 ]. The Swedish legislation might well be amended in the near future, to close the gaps mentioned in the last section. If the Government Bill 2003/04:148 on stem cell research is enacted, it will come into force on the 1st of January 2005. This Bill seeks to extend the existing legislation to make it clear that RC using the somatic nuclear transfer is encompassed and to explicitly allow somatic nuclear transfer as a way of creating embryos for non-reproductive purposes. Thus, Sweden is likely to join Belgium and the UK in permitting NRC by legislation. International initiatives i) European Convention The European Convention on Human Rights and Biomedicine has now been signed by 31 of the 45 member States of the Council of Europe, of which 15 have also ratified the Convention [ 24 ]. It has not been signed by any of the non-member participants (which include Australia, Canada, the Holy See, Mexico, and the US). While this Convention does not specifically address human cloning, a number of its provisions have implications for cloning. Article 18(2) of the Convention prohibits the "creation of human embryos for research purposes". The phrase "human embryos" is not defined by the Convention and subsequent negotiations of the working party on the protection of the human embryo and fetus appear to have failed to reach agreement on this and other issues. This provision only prohibits NRC if it captures the creation of all functional human embryos for research. While the Convention makes provision for referrals of questions of interpretation to the European Court of Human Rights (Article 29), referral is unlikely because the Convention arguably leaves such decisions to the discretion of individual States. The Strasbourg court itself allows individual States a wide discretion (known as the 'margin of appreciation') in controversial policy areas. The court has, for example, adopted this approach when considering whether the fetus is included in the provision of the European Convention on Human Rights and Fundamental Freedoms that grants 'everyone' a right to life (see the latest case: Vo v France (no. 53924/00)). Moreover, before signing or ratifying the Convention on Human Rights and Biomedicine, any State could make a reservation to this provision insofar as it is inconsistent with their pre-existing law (Article 36). This is what we would expect the UK to do if it eventually signs the Convention. Whether RC is implicitly prohibited by the Convention is more controversial. Those who hold that cloning violates human dignity will no doubt point to Article 1, which requires parties to the Convention to "protect the dignity and identity of all human beings". This seems tenuous. There is, however, an Additional Protocol on the Prohibition of Cloning Human Beings [ 25 ]. Article 1 of the Additional Protocol declares that, Any intervention seeking to create a human being genetically identical to another human being, whether living or dead, is prohibited. Since "genetically identical" is defined, under Article 1(2), as "sharing with another the same nuclear gene set", use of the Dolly technique on humans is included within this prohibition. This provision clearly captures RC. What is more controversial is whether it covers NRC. To foreclose this possibility, when the Dutch government signed the Protocol it added an interpretative statement declaring that it "interprets the term "human beings" as referring exclusively to a human individual, ie a human being who has been born". This interpretative statement is arguably unnecessary, because, in the absence of a definition of human being in the Convention itself, States are free to interpret this provision in accordance with their own national policy. The impact of these international instruments is particularly important with regard to the three countries that have ratified both: Portugal (which has no legislation), Greece (whose legislation only explicitly prohibits RC), and Spain (which has comprehensive legislation in this area). In Greece, the Explanatory Memorandum to the legislation declares that ' [i]t could be...that therapeutic cloning is permitted exactly as in Article 1 paragraph 1 of the Additional Protocol on Cloning'. We understand that conservative opinion is of the view that this interpretation is in conflict with Article 18(2) of the Convention itself. However, neither the Convention nor the Protocol on Cloning provide any sanctions for violation. ii) United Nations The United Nations' struggle to agree on a cloning treaty exemplifies both the variation of approaches and the challenges associated with seeking consensus in a morally contested area [ 26 ]. In December 2001, the UN General Assembly established an Ad Hoc Committee to consider "the elaboration of an international convention against the reproductive cloning of human beings" [ 27 ]. Since that time, a number of treaty proposals have been considered. A proposal by France and Germany, for example, recommended a narrow ban on RC only, leaving NRC for future debate [ 28 ]. A second proposal supported by Spain and the US, argued for a comprehensive ban on cloning, including NRC [ 29 ]. The most recent proposal, which was put forward by Costa Rica, would require states to establish criminal offences for all human cloning, including NRC [ 30 ]. There has, however, been little consensus on how to proceed. Though all countries agree that RC should be banned, there is deep division regarding NRC. Neither the Ad Hoc cloning committee nor the UN's Legal Committee could reach a consensus on which proposal to support and bring before the General Assembly. In November 2003 the Legal Committee voted (80-79) to recommend a two-year deferral on a General Assembly decision – a compromise that was put forward and supported by most of the members of the Organization of the Islamic Conference. This decision was largely seen as a victory for those countries supporting a more permissive approach to cloning policy [ 31 ]. Indeed, some viewed the two year delay as an ideal opportunity for the scientific community to promote the value of NRC [ 32 ]. However, in response to pressure from those countries seeking a comprehensive ban, the General Assembly came to yet another compromise. In January 2004 the General Assembly overturned the Legal Committee's recommendation and supported a one year delay on the debate over the cloning treaty. This October, the General Assembly re-opened the debate, again with no apparent compromise from either camp [ 33 ]. The fact that the deep division at the UN is primarily about NRC reflects the lack of any consistent approach to cloning policy. For example, one would expect an emerging trend toward the banning of NRC, as suggested in the quote by Senator Morin, to be reflected in the building of consensus or, at least, a degree of flexibility at the UN General Assembly. Ethical considerations Few areas of regulation are as evidentially driven by ethical views as the regulation of cloning and cloning research. This is not the place for in-depth analysis of the underlying debate. Elsewhere we have both argued that existing regulatory attempts to prohibit RC have rarely been underpinned by thoughtful exposition of underlying ethical principles [ 5 , 10 , 34 ]. Policy statements frequently rely on claims that are tautologous, under specified, poorly considered, or a combination of these things. Our claim here is more modest. In this section we seek to show why attempts to understand the ethical basis of the existing law cannot focus solely on the existing regulatory outcomes. And, of course, the regulatory outcome does not, necessarily, represent a jurisdictional consensus on the central ethical issues. Lawyers rarely look to regulatory outcome to understand ethical debates. Unfortunately, in the area of cloning many commentators do that very thing. As our starting quotation demonstrates, politicians and commentators are all too ready to find support for their ethical views in regulatory positions adopted elsewhere. There are, however, varying levels of ethical agreement. Agreement on the appropriate regulatory position does not imply agreement on the underlying ethical principles. Consider the relationship between ethical positions on the moral status (or dignity) of the cloned embryo and NRC. The cloned embryo could be considered to have full, no, or limited moral status [ 18 , 35 ]. The full status position would grant the embryo the same level of moral duties as you or I. The no status position would grant the embryo no more status or dignity than your hair or nails. The limited status position would grant the embryo a fixed or gradualist level of intrinsic moral value between these two extremes. The full status position will require the prohibition of NRC (destructive use of embryos is considered murder) and the no status position will usually require NRC to be permitted (unless such an approach will interfere with the moral interests of those who do matter). The limited status position is, however, potentially compatible with either regulatory position, depending on the particular status given to the early embryo and the weight given to potential benefits of NRC. It follows that the fact that the regulatory position permits or prohibits NRC does not do even tell us what status the embryo is considered to have. This is further complicated by the fact that supporters of the no or limited status position might be prepared to accept more restrictions than are strictly required by their position to protect a more important moral goal. Only supporters of the full status position cannot coherently make such pragmatic compromises [ 18 ]. Similarly, the existing positions on RC could be supported by radically different ethical views. Prohibitions could be supported by those who hold that RC is absolutely wrong (eg always violates human dignity) and by those who hold that cloning at present would be wrong. Even time-limited prohibition does not enable us to discern whether it is underpinned by, for example, the view that RC is wrong because of current safety issues or the view that RC is not wrong but the most effective way to get there is by initially prohibiting it. In sum, the majority of regulatory outcomes could be coherently explained by reference to one or more underlying ethical positions. Thus, similar or even identical regulatory outcomes imply less by way of ethical agreement than some may be inclined to believe. Conclusion Cloning laws and policies are far from uniform across the globe and the legal position in some countries remains uncertain. This will give little comfort to scientists and policy makers hoping to gain clear direction from the international position. For the time being at least, policymakers must accept the reality of international "dissensus" and scientists wishing to undertaken research on NRC are best advised to consider conducting their research in only a handful of countries. Even where there is agreement as to the regulatory outcome, policy-makers should not confuse this with agreement on underlying ethical principles. Like many topics concerning the developing genetic and reproductive technologies, cloning remains controversial. Competing interests The author(s) declare that they have no competing interests. Authors' contribution Both authors contributed to the original concept for the paper, the writing and revision of the manuscript and the analysis of the law. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544897.xml
524187	Role of primary surgery in advanced ovarian cancer	Background Major issues in surgery for advanced ovarian cancer remain unresolved. Existing treatment guidelines are supported by a few published reports and fewer prospective randomized clinical trials. Methods We reviewed published reports on primary surgical treatment, surgical expertise, inadequate primary surgery/quality assurance, neoadjuvant chemotherapy, interval debulking, and surgical prognostic factors in advanced ovarian cancer to help resolve outstanding issues. Results The aim of primary surgery is a well-planned and complete intervention with optimal staging and surgery. Surgical debulking is worthwhile as there are further effective treatments available to control unresectable residual disease. Patients of gynecologic oncology specialist surgeons have better survival rates. This may reflect a working 'culture' rather than better technical skills. One major problem though, is that despite pleas to restrict surgery to experienced surgeons, specialist centers are often left to cope with the results of inadequate primary surgical resections. Patients with primary chemotherapy or those who have had suboptimal debulking may benefit from interval debulking. A proposal for a better classification of residual tumor is given. Conclusions Optimal surgical interventions have definite role to play in advanced ovarian cancers. Improvements in surgical treatment in the general population will probably improve patients' survival when coupled with improvements in current chemotherapeutic approaches.	Background The Fédération Internationale de Gynécologie et d'Obstétrigue (FIGO) classifies ovarian carcinoma in stage I to IV [ 1 , 2 ]. Stage I has been defined as growth limited to the ovaries; stage II as growth involving one or both ovaries with pelvic extension; stage III as tumor involving one or both ovaries with peritoneal implants, and outside the pelvis and/or positive retroperitoneal or inguinal nodes and stage IV as having distant metastasis [ 1 , 2 ]. Tumors in stages I and II are generally considered to represent early disease, while stages III and IV evince late or advanced disease [ 3 , 4 ]. The strong prognostic value of the FIGO classification system has been proved in number of studies [ 5 ]. Unfortunately, most ovarian carcinomas are detected only when they are advanced. Results of studies evaluating screening by tumor markers (a raised CA125 value) and/or ultrasonography to detect early disease are not clear [ 6 , 7 ]. Ultrasonography may become an increasingly important tool as it has been associated with higher detection rates in early stage disease and in patients with a genetic predisposition to tumor [ 8 - 11 ]. However, use of proteomics will perhaps identify more ovarian carcinomas at early stages in the future [ 12 ]. Though controversial till 70s, surgery is now recognized an integral part of the treatment armamentarium in advanced ovarian carcinoma. Aure et al ., [ 13 ] presented convincing evidence that extensive tumor removal resulted in better survival even in advanced stage disease and introduced the idea of primary tumor debulking surgery. The value of primary debulking surgery was confirmed and its theoretical background was elucidated by Griffith and Fuller [ 14 ]. Subsequent work showed that debulking surgery improves an adverse vegetative function and nutritional problems such as loss of appetite and nausea [ 15 , 16 ]. It was also suggested that primary debulking surgery removes therapy-resistant tumor cells and increases the number of proliferating tumor cells (the Gompertzian phenomenon), which makes these cells more susceptible to subsequent chemotherapy [ 17 - 19 ]. These early hypotheses have partly been confirmed by the finding of increased postoperative tumor proliferation rates in patients after surgery [ 20 ]. Ongoing discussions about quality assurance and guideline-based therapy had helped to foster the impression that the main issues in treating ovarian cancer have been resolved and that the value of each procedure involved has been supported by high levels of scientific evidence [ 21 - 23 ]. Closer inspection reveals that it is not true. Only a few treatment guidelines are supported by published reports, and even fewer by prospective randomized clinical trials. However, we strongly believe the value of retrospective studies is greatly underestimated. Recent analyses show that the treatment effects assessed by observational studies do not greatly differ in magnitude or quality from those published in randomized, controlled trials [ 24 , 25 ]. Furthermore, biases created by the selection criteria inherent in prospective randomized trials are frequently ignored. Thus, in this article we will concentrate on looking more closely at several issues in surgical treatment, their effects and importance in relation to outcome in advance ovarian cancer. Primary surgical treatment The utility of primary surgery for advanced ovarian cancer is well established. Its aim should be a well-planned, extensive and complete intervention. Thus, no facility should offer surgery for patients with ovarian cancer if adequate standards of care cannot be met. The optimal preparation of patients for surgery is very important. Patients must be in a position to give fully informed consent to any additional surgical procedures found necessary during the operation. They should also undergo colonic lavage, which will provide the surgeon with better access to the lymph nodes and reduce risks in cases where intestinal surgery is undertaken. Where cytological evaluation of peritoneal fluid aspired preoperatively, a raised concentration of serum CA125, or ultrasound results indicate advanced malignancy, the patient should be transferred to a specialist surgeon (see below). Many cases where the staging is not clear, laparoscopy appears to be good tool for obtaining a definitive histological diagnosis in advanced ovarian cancer and helps in planning the best surgical approach. Laparoscopic staging, in particular, can give a clear view of the extent of surgery required and the difficulties that may be expected, and it may be helpful in selecting patients for primary (neoadjuvant) chemotherapy [ 26 ]. Video recording can document the findings during laparoscopy and can be used subsequently by the surgeon to plan optimal debulking surgery. However, laparoscopic surgery of any suspicious adnexal mass is not encouraged unless the risk of capsular rupture and tumor spill is minimized by the use of endobags [ 27 ]. No cystic mass which is >10 cm in diameter and/or adherent to the lateral pelvic wall should be removed laparoscopically [ 28 ]. The fear due to some in-vitro and animal studies that showed carbon dioxide pneumoperitoneum has adverse effects on outcomes is probably unfounded, as a recent analysis from second-look laparoscopies showed no influence of pneumoperitoneum on overall survival [ 29 ]. Certainly, more data on this issue is required. Since laparoscopy may increase tumor growth rates, delays between laparoscopy and definitive surgery should be avoided [ 30 ]. Although this view has not yet been supported by any other study, we believe that the time between the suspected diagnosis of advanced ovarian cancer and surgery should be kept as short as possible. Delays may result in a higher preoperative tumor mass which has been identified as an adverse prognostic factor [ 31 ]. Surgical staging Tumor stage is one of the primary prognostic factors. Appropriate staging is vitally important for effective postoperative therapeutic decision-making. Patients who have been accurately staged as stage I may not require adjuvant chemotherapy [ 32 , 33 ]. Requirements for appropriate staging after total abdominal hysterectomy and bilateral salpingo-oophorectomy include multiple cytological washings, random biopsies from the peritoneum and the diaphragm, omentectomy and lymphadenectomy. The value of peritoneal cytology is supported by prospective studies [ 34 ]. There are several issues surrounding lymphadenectomy, and these are discussed later. In two studies, optimal staging resulted in 30% to 50% of the patients being reclassified to a higher stage – a fact which has implications for subsequent treatment [ 35 , 36 ]. A classification system for determining the quality of surgical staging was introduced recently and is shown in Table 1 [ 33 ]. However, it may be only helpful for comparisons of older studies since optimal staging is a prerequisite of later therapeutic decisions. Table 1 Surgical quality categories for staging of ovarian carcinomas (based on Trimbos et al. 2003) Category of surgical quality Staging procedures included Optimal - Inspection and palpation of all peritoneal surfaces; biopsies of any suspect lesion for metastasis; peritoneal washings; infra-colic omentectomy; blind biopsies of the right diaphragm and right and left para-colic gutter, pelvic side-walls of the ovarian fossa, of the bladder peritoneum and of the cul-de-sac and sampling of iliac and para-aortic lymph nodes Modified - Everything between optimal and minimal staging Minimal - Inspection and palpation of all peritoneal surfaces and the retroperitoneal area; biopsies of any suspect lesions for metastasis; peritoneal washing; infracolic omentectomy Inadequate - Less than minimal staging but at least careful inspection and palpation of all peritoneal surfaces and the retroperitoneal area; biopsies of any suspect lesion for metastasis Extent of surgery In addition to the staging procedures mentioned earlier, optimal surgical treatment for ovarian cancer comprises tumor removal; removal of remaining ovaries, uterus, and fallopian tubes, omentectomy, and radical para aortic and pelvic lymphadenctomy [ 3 , 37 ]. The German national treatment guidelines recommend a simultaneous appendectomy and removal of the cul de sac over the peritoneum of the small pelvis [ 38 ]. Since removal of all grossly visible tumor is considered crucial for long-term survival, surgery should be extended to include hemicolectomy, splenectomy and stripping of the peritoneal reflection of the diaphragm when the tumor masses infiltrate the entire abdominal cavity, the colon, the diaphragm or other structures respectively. Although the reasoning behind performing these measures seems convincing, only lymphadenectomy has been partly evaluated in a prospective, randomized trial. The previously held belief that mere palpation of lymph nodes is sufficient to gauge nodal status was refuted in this study [ 39 ]. Lymphadenectomy plays a triple role in the treatment of ovarian cancer. First, it is of diagnostic value since tumors of apparently early stage show nodal involvement in about 20% to 40% of the cases [ 40 ], If found positive, the tumor must be classified as stage IIIc. Secondly, lymphadenectomy is of immense prognostic value. Most importantly, lymphadenectomy may also have a therapeutic effect as retrospective studies comparing lymphadenectomy with no lymphadenectomy reported a survival benefits with this procedure [ 41 - 43 ]. The data on lymphadenectomy is however conflicting with one study showing that the patients with stage III disease (tumor residuals >2 cm) that has been debulked suboptimally do not benefit from lymphadenectomy [ 44 ]. Other workers report no benefit even if the residual tumor size is smaller (1 cm) [ 45 ]. Though not fully published, the only prospective, randomized trial shows that systematic lymphadenectomy did not result in better survival compared to selective lymphadenectomy [ 46 ]. Mainly based on the retrospective findings current views on treating stage III disease suggest: systematic lymphadenectomy in cases of residual tumors <1 cm, nodal debulking only where tumors are larger than intra-abdominal residuals, and nodal sampling in stage IV disease with pleural effusions only [ 47 ]. To the best of our knowledge, there has been no study testing the benefit of hysterectomy or omentectomy. However, the concurrent incidence of endometrial carcinoma in 10% to 25 % of patients, or its precursors in about 30% to 50% of all ovarian cancers justifies this procedure [ 48 , 49 ]. Optimal debulking Ovarian cancer is one of the tumors where surgical debulking is considered worthwhile. This is due to availability of further effective treatments that are available to control the unresectable residual disease. As early as 1934, Meigs suggested that maximum cytoreductive surgery was beneficial [ 50 ]. Many years later, in 1968, Munnell followed this idea and proposed the idea of 'maximum surgical effort' [ 51 ]. He distinguished between definitive surgery, partial removal of the tumor and biopsy only. Since partial removal covers a wide range of interventions that requires varying amount of efforts, optimal debulking was distinguished from suboptimal debulking. Although there is no generally accepted definition, most early studies considered a residual tumor size of <2 cm as optimal [ 52 ]. In a more recent survey among gynecological oncologists from United States of America (USA), 12% of the responders defined optimal debulking surgery as no visible tumor residuals, while 14% described it as residual tumor masses less than 0.5 cm. However, 61% chose a 1 cm threshold and 13% considered a tumor of 1.5 cm to 2.0 cm as optimal [ 53 ]. Comparative analysis of diameters of various residual diseases has shown that there exists some sort of a threshold at 2 cm, above which no significant differences in survival can be found. In contrast, subset analyses of smaller diameters in residual disease show improved patient prognosis [ 54 ]. This variation in the interpretation of thresholds with prognostic impact calls for a commonly accepted definition (see concluding remarks) and more controlled trials, that need to be non-randomized as it will not be ethically possible to leave some tumor behind. The results of an earlier meta analysis on cytoreductive surgery might have been flawed not only due to absence of clear definitions but also due to the combined effects of subsequent chemotherapy [ 52 ]. In this study, the then novel, platinum-containing chemotherapy had a stronger impact on survival than cytoreductive surgery. A recent and otherwise comparable meta analysis however, confirms the greater survival benefit of patients undergoing maximum cytoreduction [ 55 ]. Another interesting study stated that optimal cytoreduction means no visible residual tumor [ 31 ]. It has been further shown that the volume of the residual tumor and the success of subsequent chemotherapy are interdependent [ 56 ]. Expertise of gynecological oncology surgeons Based on available evidence it is generally accepted that the experience and technical expertise of a surgeon are important prognostic factors. Comparisons of overall survival in patients treated by gynecological oncology, gynecologists and general surgeons have shown that patients treated by surgeons trained in gynecology (gynecological oncology) have a significantly better prognosis [ 57 , 58 ]. This finding may not reflect primarily on the technical skills of these surgeons but rather reflection the 'environment' in which they work – where views and thoughts on the biology of advanced tumors are freely shared and patients are often treated by a team rather then individuals. As for surgeons there are only select patients with uncommon neoplasms like gastrinomas, glucagonomas, stomatostatinomas, and VIPomas, who profit from debulking surgery with particular reference to prevent deleterious hormonal side-effects [ 59 ]. It is expected that new chemotherapy and immunotherapeutical approaches will probably lead to a re-evaluation of debulking surgery as a complementary approach [ 60 - 62 ]. Till such time where definite evidence is available, it is strongly recommended that all patients should be treated by a gynec-oncologist. There had been constant calls to regionalize specialist surgery, however, no studies have yet shown better survival in patients treated by 'high volume' operators or such specialists [ 63 , 64 ]. A recent study on quality control from Hesse, Germany, showed striking deficiencies even at central-referral hospitals [ 23 ]. Treatment by a multidisciplinary team of specialists, has been shown to increase patients' chances of survival without any disputes [ 65 , 66 ]. Patients probably benefit most from being treated in centers which promote excellent scientific exchange, and continuous education and self-evaluation among surgeons besides providing multidisciplinary approach to management. Coping with inadequate primary surgery Surgical treatment of advanced ovarian cancer is one of the most demanding procedures in gynecological surgery. Despite repeated requests to restrict surgery to experienced surgeons, considerable numbers of patients are still operated by others. A population based study from Germany showed that omentectomies were performed in about 50% of all cases of ovarian cancer and lymphadenectomy were carried out only in 30% [ 22 ]. Another study from USA showed that only about half of the patients receive 'standard' care [ 68 ]. In spite of the establishment of gynecological oncology as a specialty in the USA, fewer than half of the patients were originally seen by such a specialist [ 68 ]. More over the terminologies like "standard" are not defined well. The situation with regard to specialism depends strongly on the medical infrastructure, and varies from country to country and region to region. However, the problem of inadequate primary surgery is real, and coping with it is a frequent task in specialist centers even in developed countries. The question is what should be done for the patient concerned? Interestingly, this is something that cannot be found in textbooks [ 3 , 69 , 70 ]. Some of the literature suggest re-laparotomy by experienced surgeons to achieve reductions in all possible tumor mass [ 71 , 72 ] however, there is no evidence to support this strategy. In general, one of two situations occurs. First patients present with no evidence of macroscopic tumor residuals but staging procedures and/or operative measures were omitted. Computed tomography (CT) or Magnetic resonance imaging (MRI) to identify enlarged lymph nodes or possible residual tumors in these situations may help to decide on the need for second surgery. The belief that tumor cells in retroperitoneal lymph nodes are better able to survive chemotherapy is supported by low cytotoxic drug concentrations in these [ 73 ]. Therefore, it is reasonable to consider enlarged lymph nodes as a decisive factor favoring a direct surgical approach. It is interesting to note that in endometrial carcinomas, a clinically negative omentum was also found to be histologically negative in most cases (sensitivity 89%) [ 74 ]. Patients with residual tumor mass have to be evaluated to determine if it is possible to achieve no residual tumor or microscopic residual tumor by immediate secondary surgery. Although immediate laparotomy seems to be a good idea, the limited capacity for surgery at specialist departments and delays in having the surgery are to be considered. The peritoneum shows inflammation shortly after surgery reaching a high about 7 to 14 days afterwards [ 75 ]. Surgery at this time is considered far more complicated and may result in higher blood loss and greater risk of injury to neighboring abdominal organs [ 76 ]. However, waiting for the inflammatory processes to resolve will give the tumor further time to proliferate [ 20 ]. Therefore, interval debulking surgery after three courses of chemotherapy should be considered as an appropriate alternative. Neoadjuvant chemotherapy Disease spread >2 cm to the spleen, diaphragm, liver surface, mesentery, or gallbladder is generally believed to be inoperable. However, even these patients may often undergo effective debulking procedures [ 26 , 77 ]. As mentioned earlier, laparoscopy can be used to reach decisions on surgery. The only problem with laparoscopy in combination with neoadjuvant chemotherapy is a 30% rate of port site metastasis, which is believed to be a result of the pneumoperitoneum procedure created for laparoscopy [ 78 ]. These metastases should be excised at the time of any subsequent surgery [ 79 ]. An analysis of several retrospective studies on neoadjuvant chemotherapy showed that there are no good reasons to assume that this approach is associated with a poorer prognosis [ 26 ]. The European Organisation for Research and Treatment of Cancer (EORTC) protocol 55971 comparing upfront tumor debulking surgery with neoadjuvant chemotherapy in patients with stage IIIc or IV disease is accruing and its results will provide better insights on this issue. Interval tumor debulking Interval debulking, is another approach to reduce tumor burden between the cycles of chemotherapy. It has been evaluated in two prospective randomized trials [ 80 , 81 ]. The EORTC study showed a clear survival advantage for interval debulking (still noted in the 2001 update), the Gynecologic Oncology Group (GOG-152) study has, as yet, failed to show any benefit from interval debulking [ 80 , 81 ]. Patients with residual tumor >1 cm received three courses of cyclophosphamide/cisplatinum in the EORTC study [ 80 ] or three courses of paclitaxel/cisplatinum in the GOG-152 study. In both studies, patients who did not respond to chemotherapy were removed from the study. Those who responded were randomized to either secondary surgery or no surgery. Afterwards, all patients received three more courses of the earlier chemotherapeutic regimen. Although there seem to be only minor differences in the design of both trials, a closer look shows that in the GOG-152 study, the number of stage IV patients was lower (6%) compared with the EORTC (21%) study, the performance status was better, and there was less residual tumor. This was due to the eligibility criteria for GOG 152 which stated that patients should have had surgery with maximal effort to resect the uterus, tubes, ovaries, omentum, and all gross residual ovarian cancer at the time of primary surgery. The questions about the benefit of interval debulking surgery remain unresolved. However, it appears that patients who have had neoadjuvant chemotherapy or suboptimal debulking may profit from this treatment, while those who have undergone primary, maximum effort surgery by a gynecological oncologist are less likely to profit from it [ 82 ]. Surgical prognostic factors Size of the residual tumor, volume of the residual disease and experience of the surgeon are important prognostic factors [ 56 , 77 ]. Among these, only few can be influenced by human intervention. Except for dose intensity of chemotherapy, recent literature indicates that the hemoglobin concentrations before chemotherapy are of prognostic value [ 83 , 84 ]. The latter may be influenced by the use of erythropoietin which has shown positive effects on survival in cervical cancer [ 86 ]. As mentioned above, the frequently used definition of <2 cm for optimal debulking is arbitrary since every further reduction in the size of residual tumor improves the prognosis [ 54 ]. Thus, each threshold between 0 and 2 cm will have its own prognostic relevance. While the criterion 'diameter of residual tumor' reflects tumor cell hypoxia and reduces the pool of proliferating tumor cells susceptible to chemotherapy, the criterion 'residual tumor volume' alludes to the removal of therapy-resistant tumor cells, which are believed to be responsible for early recurrences. A comparison of both criteria in relation to their prognostic impact has shown that residual tumor volume is of greater importance [ 56 ]. As shown in subgroup analyses of the intergroup trial confirming the results of the GOG-111 study, a possibly superior chemotherapeutic regimen containing taxanes cannot compensate for the tumor left behind after primary surgery [ 19 ]. Future trends It is difficult to assess the future role of surgery in advanced ovarian cancer. Neoadjuvant chemotherapy may become more important. However, as the tumor debulking surgery works, except in stage IV patients with solid distant metastasis, it may be worth trying combined ultrasound-guided laser interstitial thermotherapy for non-resectable liver metastasis with conventional debulking surgery [ 87 ]. Apart from technical innovations, quality control, quality assurance and documentation of patient outcomes after surgery will probably play major parts in treatment improvements. Conclusion Numerous studies have analyzed the effects of various kind of chemotherapy in ovarian cancer. In contrast, only a few prospective randomized studies have focused on surgical issues in this type of tumor [ 46 , 80 , 81 ]. This lack of surgical trials has probably contributed to inhomogenous definitions regarding the terminology of surgical interventions and surgical stages (early compared with late) and the classification of operative success in general. In future, we must aim to ensure that all patients are treated along the generally accepted guidelines and receive optimal debulking surgery which leaves only microscopically detectable residual tumor as shown in number of studies; it is certainly unethical at present to evaluate this procedure in randomized clinical trials (see Introduction). Other then this, there are many other relevant issues which need to be resolved or clarified with special reference to neoadjuvant chemotherapy, interval debulking, the surgeon's training, and inadequate primary treatment. It would certainly be very helpful to demonstrate clearly the consequences of what is supposed to be an inadequate treatment. The simple dichotomization of FIGO stages to early or late does not correspond to any diagnostic, biological or therapeutic advantage. We believe that it is far more reasonable to consider stage I alone as early disease (perhaps even only stage Ia and b), stages II and III (perhaps even stage IV with pleural effusions) as intermediate disease, and stage IV with organ metastasis as advanced disease. Such a classification would follow current views on treatment, since early ovarian carcinomas are treated primarily by surgery (eventually fertility-sparing) and adjuvant chemotherapy in cases of increased risk, intermediate ones by surgery and routine chemotherapy, and advanced disease by chemotherapy only. Thus far, results of studies on stage IV patients with organ metastases are inconsistent regarding the benefit of surgery. This is another issue to be resolved [ 88 ]. In this respect, FIGO may find subdividing stage IV into stage IVa (pleural effusions) and IVb (organ metastasis) worthwhile. Disappointingly, the Tumor-Nodes-Metastasis (TNM) classification follows the FIGO system and violates its own principles by not accepting distant peritoneal metastasis as a natural indicator of primary tumor size but also by summarizing a nodal-positive disease stage as T3c (corresponding to FIGO IIIC) regardless of intra-abdominal findings. This inconsistency has already created curious confusions in current research. Nodal involvement has been shown to impair prognosis, however, smaller intra-abdominal tumors with nodal involvement (presented as stage I to IIIb disease but, by definition, all stage IIIC) show a significantly better prognosis than extensive intra-abdominal tumor masses (again stage IIIC) [ 89 ]. Furthermore, residual disease should be properly defined. The most rational approach is to regard microscopic residuals as optimal. Case series claim that experienced gynecologic-oncologic surgeons can clear up to 85% of patients in the unfavorable subgroups (FIGO stage IIIc and IV) of all visible tumor, leading to an extraordinarily high five year survival rate of about 50% [ 77 ]. As discussed (see surgical prognostic factors), a good definition of residual tumor would include aspects of both residual tumor size and volume. A proposal is made in Table 2 . Table 2 Surgical documentation of residual tumor after debulking of ovarian carcinomas Residual tumor status * Maximum diameter of residual tumor Maximum total volume of residual tumor Optimal Microscopic No visible tumor Minimal < 1 cm ≤ 10 cm 3 Intermediate 1 – 2 cm > 10 cm 3 but ≤ 100 cm 3 Gross > 2 cm > 100 cm 3 *To assign residual tumor to a certain status, both criteria, diameter and volume, have to be fulfilled. Otherwise the next lower category should be used. In summary, more attention need be paid to surgery for advanced ovarian cancer. These include necessary improvements in treatment in the general population, uniform definitions and terminology, and increasing number of surgical clinical trials. Extrapolating from the results of truly optimal ovarian cancer surgery, we believe that improvements in surgery will lead to better patient survival than improvements in current chemotherapeutic approaches [ 55 , 90 , 91 ]. Competing interests The authors declare that they have no competing interests. Authors' contributions KM and FEF both participated equally in literature search, conceptualization and preparation of the manuscript. Both authors have read the manuscript and approve it for publication.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524187.xml
529272	Recruiting pregnant smokers for a placebo-randomised controlled trial of nicotine replacement therapy	Background Smoking in pregnancy is a public health problem and effective methods for reducing this are required. Although nicotine replacement therapy (NRT) is effective for smoking cessation in non-pregnant people, there is no direct evidence concerning its effectiveness in pregnancy. Despite this, clinical guidelines recommend the cautious use of NRT during pregnancy. Randomised controlled trials are needed to determine the safety and efficacy of NRT when used by pregnant women for smoking cessation, but the feasibility of recruiting women to such trials is unknown. Consequently, in this study we aimed to determine i) the feasibility of recruiting women to a RCT of NRT in pregnancy as they attend hospital antenatal ultrasound examinations, ii) the proportion of such women who are eligible for and interested in trial enrolment and iii) research staff perceptions of how one method of trial recruitment could be improved. Methods During a one month period, all women attending for antenatal ultrasound examination in an English teaching hospital were asked to complete a questionnaire which determined their eligibility to enrol in a proposed placebo controlled randomised trial investigating the effectiveness of NRT in pregnancy. Women who were eligible to participate were asked whether they would do so and those who accepted enrolment were offered an appointment with a smoking cessation advisor. Results Over 99% (851/858) of women agreed to complete a questionnaire about smoking habits whilst waiting for ultrasound examinations. 10.3% (88/851) of women attending for antenatal ultrasound fitted eligibility criteria for a proposed RCT of NRT in pregnancy, but only 3.6% [(31/851), 95% CI, 2.4 to 4.9%] indicated on the questionnaire that they would like to take part in a study involving randomisation to placebo or active patches. Researchers offered trial enrolment to 26 of these 31 women and 96% (25) accepted. Staff recruiting women believed that trial recruitment would be maximised if women attending the ultrasound department knew about trial recruitment before attending and greater staff resources were made available for this. It was also perceived that women generally under-reported the amount they smoked on questionnaires completed whilst waiting in ultrasound department areas. Conclusions It is feasible to recruit women for a trial of NRT in pregnancy as they wait for antenatal ultrasound examinations. Using similar recruitment methods, researchers can expect to recruit between 24 and 49 women per 1000 approached.	Background Smoking harms unborn children causing intra-uterine growth restriction, preterm birth, miscarriage and perinatal death [ 1 , 2 ]. Despite this over a quarter of pregnant women in the UK smoke [ 3 ] and although pregnancy motivates a minority of women to stop smoking for at least part of their pregnancy, most start again after delivery [ 3 ]. Reducing smoking in pregnancy is, therefore, a health priority and effective methods for promoting smoking cessation in pregnancy are needed. Behavioural support is effective for smoking cessation in pregnancy, [ 4 ] but drug treatment is not generally used because of concerns that this may harm the fetus [ 5 ]. Outside of pregnancy, however, drug therapy can be effective and nicotine replacement therapy (NRT) increases quit rates of smoking cessation interventions it accompanies by 1.5 to 2-fold [ 6 ]. Unfortunately, the efficacy and safety of NRT in pregnancy are unknown. Two trials conducted in pregnant women have shown NRT patches to have no greater effect on smoking cessation than placebo [ 7 , 8 ]. but neither trial has been large enough to be definitive. Additionally, nicotine is metabolised more quickly in pregnancy [ 9 ] and this could reduce the efficacy of NRT, as conventional doses will provide less nicotine substitution. Consequently, for NRT to be effective in pregnancy, higher doses might be needed but this raises questions of safety because nicotine is a recognised fetal toxin [ 10 ] and higher doses could increase the risk of any fetal damage occurring. Any such risk is difficult to quantify, though, because there is little human-subject evidence on the effects of nicotine (or NRT) in pregnancy and most evidence about the impact of nicotine in pregnancy is derived from animal studies [ 10 ]. In humans, nicotine is known to increase maternal blood pressure and heart rate but to have lesser effects on the fetal heart rate and these changes are less pronounced than those caused by smoking [ 10 ]. More encouragingly, the one completed trial of NRT in pregnancy [ 7 ] found babies born to women in the NRT treatment group had significantly higher birth weight than those treated with placebo, suggesting that intrauterine growth restriction caused by smoking is not attributable to nicotine. Further evidence on the safety of nicotine in pregnancy would help to inform clinicians' decisions about prescribing NRT in pregnancy. Despite uncertainties about the effects of NRT in pregnancy, there is an expert consensus that NRT use is safer than smoking [ 11 ] which is reflected in clinical guidelines [ 5 , 12 ] and other advice to clinicians [ 13 ]. NRT use in pregnancy is, therefore, being encouraged in the absence of any direct evidence for it's effectiveness or safety and placebo-controlled RCTs are needed to resolve these issues. In pregnancy, women are generally advised to avoid unnecessary drugs [ 14 ] and it is likely, therefore, that women's reluctance to use drugs (including NRT, which contains a known fetal toxin [ 10 ]) might hinder recruitment to RCTs of NRT in pregnancy. Consequently, this paper describes a pilot study which aimed to determine: i) the feasibility of recruiting women to an RCT of NRT in pregnancy as they attended hospital antenatal ultrasound examinations, ii) the proportion of pregnant women attending such clinics who are eligible for enrolment and interested in participation in such a trial and iii) research staff perceptions of how one method of recruitment could be improved for use in a definitive RCT. Methods Ethical approval was granted by the Nottingham Research Ethics Committee 2. For one month, one of three research assistants (RAs) was based in the Nottingham City Hospital antenatal ultrasound department for all scanning sessions. All women attending for ultrasound scans were approached by an RA and asked to fill in a self-completion questionnaire (see appendix) which asked their number of weeks gestation and whether or not they smoked at all. Respondents who answered that they smoked cigarettes or tobacco were then asked a series of questions about their smoking behaviour to ascertain whether or not they satisfied eligibility criteria for a proposed RCT (see Figure 2 ). Those smokers who were at an appropriate gestation, who smoked heavily enough for entry to the RCT and who were interested in stopping smoking completed the whole questionnaire. Before the final questionnaire item, they read brief information about the use of nicotine replacement therapy in pregnancy (which was on the questionnaire). The final item then asked women whether or not they would like to take part in a study which investigated the effectiveness of NRT in pregnancy. Those who did were subsequently offered enrolment into the study by the RA and their responses were noted. The enrolment offer included an explanation that no randomised study of NRT was currently taking place but that this was possible in the near future (which, at that time, the authors believed to be true). Women who agreed to enrolment were aware that they might participate in a study that would involve them being randomised to either active or inactive patches and, although nicotine was potentially harmful, NRT use was believed to be safer than smoking. RAs arranged appointments with the local NHS smoking cessation service [ 15 ] for those women who accepted the offer of enrolment and, if they attended these, women received counselling from midwives who specialised in smoking cessation counselling for pregnant women. Enrolment criteria for the proposed trial (Figure 2 ) were constructed to ensure that only women who were addicted to nicotine were likely to be enrolled in the study. It was important to prevent any woman from entering a trial if it were possible that she might receive more nicotine via NRT dispensed in the trial than she otherwise would have received by smoking [ 11 ]. The antenatal ultrasound clinic setting was used because almost all pregnant women in the UK have an ultrasound scan performed between the end of the first and the middle of the second trimester of pregnancy. By recruiting in this setting, we were able to systematically approach all pregnant women residing in the local area who were at an appropriate stage of gestation and who were likely to deliver in the same hospital. When the pilot was finished, the views of RAs and collaborating staff from the local NHS smoking cessation service were sought at a meeting. We asked their opinions of the utility of our method for detecting smokers (i.e. the questionnaire) and the practicality of recruitment within the ultrasound clinic setting. Where staff had criticisms, they were encouraged to suggest improvements in procedures. Key points made during this meeting were noted and issues described in the results section are those on which there was consensus. Sample size and data analysis We knew from routinely-collected clinic statistics that over 800 women would attend for antenatal ultrasound scans in a four week period and around 200 of these (25%) would smoke. We hypothesised that 20% of smokers (i.e. approximately 40 or 5% of clinic attenders) would agree to trial randomisation. We believed this a reasonable assumption because in a survey of women attending ante-natal clinics, Ussher found that, amongst women who smoked 10 or more cigarettes daily and who were interested in stopping smoking; 56% were also interested in using NRT [ 16 ]. Consequently, if our survey was conducted for one month, we calculated that would be able to detect the proportion of clinic attenders agreeing to randomisation with 95% CIs of 1–2%. All data was entered into an SPSS database and quantitative findings are expressed as simple proportions of all clinic attenders, with 95% CI for the principal outcome. Results Figure 1 shows that 99.3% of women attending for antenatal ultrasound imaging during the study period consented to complete a questionnaire. Of questionnaire respondents, around 27% had smoked in the last week, but only 163 of these were within the appropriate gestation range for trial entry. The diagram shows that although 17% of attenders admitted to smoking on "at least most days", this number fell to around 13% who admitted smoking 10 cigarettes daily before they were pregnant and it fell further to 10% who admitted smoking both 10 cigarettes daily before pregnancy and 5 currently. Of these heavier smokers, 5.6% were interested in stopping smoking. Finally, 3.6% [(31) 95%CI, 2.4 to 4.9%] of women attending for ultrasound both met trial entry criteria as measured on the questionnaire and were interested in participation in a study involving NRT. RAs offered study enrolment to 26 of these 31 women and 25 (96%) accepted. The remaining 5 women left the ultrasound clinic before RAs could discuss participation with them. 21 women who agreed to the enrolment offer attended at least one appointment with the local NHS smoking cessation in pregnancy advisor. Perceptions of research and smoking cessation service staff involved in pilot There was consensus amongst RAs and NHS smoking cessation service staff that, if the following problems with study procedures were remedied, the proportion of women interested in trial participation who actually enrolled should increase: • A number of women were interested in study participation but did not have sufficient time to discuss this with the RA in the clinic. These women might have been able to make time to discuss the study if they had known about the likelihood of being approached before attending the ultrasound department. • On occasions, RAs failed to follow up women who had expressed an interest in study participation because they were busy with other duties (e.g. discussing enrolment with another woman). • A number of women who had indicated on their questionnaire that they were interested in participation left the clinic before the RA could discuss enrolment with them. The pilot study did not have the resources to contact these women afterwards. • Some women reported heavier smoking to smoking cessation advisors than they recorded on questionnaires. This apparent under-reporting of smoking behaviour may have resulted in some women who were actually eligible for study participation, not being offered study enrolment. Discussion We have demonstrated that it is feasible to recruit women to a trial of NRT in pregnancy as they attend antenatal ultrasound appointments. Although almost all women consented to complete a questionnaire enquiring about their smoking habits, only 10% of these were eligible to enrol in an RCT and less than 4% appeared likely to participate in one. Our study provides useful, original information for researchers planning trials of NRT in pregnancy. The two published trials of NRT use in pregnancy give no information about numbers of women were asked to participate but declined. One trial took two years to enrol 250 pregnant women [ 7 ] and the other recruited 30 women, but the time taken to achieve this is not reported [ 8 ]. To determine the level of interest in using NRT amongst pregnant smokers, Ussher and West conducted a telephone survey of women who had reported themselves to be smokers during a "booking" appointment (i.e. their initial antenatal appointment) [ 16 ]. They found that around one half of women who admitted to smoking at their antenatal "booking" appointment (95/177) were interested in receiving help with stopping smoking, and of those who admitted to smoking 10 or more cigarettes daily, 56% were interested in using NRT. This survey, however, was conducted amongst an ante-natal clinic population with a smoking prevalence of around 12%, which is much lower than the national average, so the generalisability of study findings to other areas is questionable. Additionally, it gave no indication of the numbers of women who might agree to randomisation which included the possibility of being allocated a placebo treatment. Nevertheless, our findings are not necessarily inconsistent with the London antenatal clinic survey. We found that approximately 35% (31/88) of women currently smoking five cigarettes daily (who also admitted to smoking 10 cigarettes daily before pregnancy) were interested in trial participation. Our findings compliment those of Ussher's survey by providing information about likely participation rates for any randomised controlled trial. Extrapolating our figure of 3.6% indicates that approximately 36 (95%CI, 24 to 49) women could be recruited annually to a RCT of NRT in pregnancy from a maternity hospital with 1000 births/pregnancies each year. Researchers planning trials of NRT in pregnancy can use this recruitment rate to inform the planning of studies. Trialists should note, however, that we have measured agreement to be randomised rather than actual randomisation. Additionally, we did not measure the numbers of exclusions amongst women who were interested in participation. Figure 2 , however, lists exclusion criteria for a RCT of NRT in pregnancy that the authors have proposed and consideration of these suggest that few pregnant women agreeing to enrolment would be excluded from using NRT on medical grounds. Finally, pilot study findings are likely to be most applicable to countries which have policies on the use of NRT in pregnancy that are similar to those employed in the UK: the proportion of women agreeing to randomisation would probably be lower in any health care system in which health professionals generally advised pregnant women against using NRT. Some modifications to the recruitment method we used might be needed to maximise trial recruitment rates. Recruitment from antenatal ultrasound waiting areas might be increased if women were sent information about the likelihood of being approached to join any trial before they attended (e.g. sending information out with appointments). Additionally, providing enough resources to ensure that a member of trial staff is always present to ask patients to join a trial is important. This would overcome the problem of missing potentially interested women when the researcher responsible for recruiting is busy enrolling other potential participants. Any recruitment method should also allow for the fact that some women who are interested in participating will not be able to wait in the clinic to discuss this further. Recruitment would, therefore, be enhanced if enough resources were provided to allow research staff to make contact with these women later, visiting them at home if necessary. The observation by smoking cessation service staff that some women admitted smoking more heavily to them than they admitted on questionnaires is consistent with the literature. Aveyard investigated the relationship between urinary cotinine levels and reported numbers of cigarettes smoked during pregnancy and concluded that, at booking, some pregnant women probably minimise the numbers of cigarettes that they report smoking [ 17 ]. Similarly, Owen and McNeil's investigation of salivary cotinine levels in pregnancy indicated that a small number of pregnant women actually conceal the fact that they smoke [ 18 ]. These research findings probably reflect the fact that, in the UK, there is a social stigma attached to smoking in pregnancy which is likely to bias questionnaire respondents against an honest disclosure of their smoking habit. Some women in our pilot may have been happier to disclose the full extent of their smoking to a supportive health professional than they were to a questionnaire distributed by a RA in an out patient waiting area. Trialists could reduce the impact that under-reporting of heaviness of smoking might have on trial recruitment by avoiding the use of questionnaires to quantify smoking habits. Questionnaires used in recruitment to trials of NRT in pregnancy might be more effective if they merely ask women to disclose whether or not they smoke and if they are interested in using NRT to stop. All who smoke and wish to stop using NRT could then be invited for further discussion of trial entry with a researcher or health professional and during this disclosure a more accurate assessment of heaviness of smoking might be obtained. Conclusions We have demonstrated that it is feasible to recruit pregnant women to a trial of nicotine replacement therapy in pregnancy in the antenatal ultrasound clinics. Using similar recruitment methods between 24 and 49 pregnant women per 1000 approached are likely to consent to participation in an RCT. RCT(s) of NRT in pregnancy are required and this data will help researchers planning such trials to determine the resources required to answer important questions concerning the effectiveness and safety of NRT in pregnancy. Competing interests TC has been paid for speaking at a conference by GlaxoSmithKline (GSK); he has also done consultancy work on one occasion for Pharmacia (now Pfizer). JB has been reimbursed by Glaxo Wellcome (now GSK) for attending 2 international conferences, has received a speaker honorarium from Glaxo Wellcome, has been the principal investigator in a clinical trial of nicotine replacement therapy funded by Pharmacia and has undertaken consultancy work for Novartis. All companies manufacture nicotine replacement products but none were involved in this study. All other authors have no conflicts of interest. Authors' contributions All authors were involved in various stages of study design. MA liaised with the NHS Stop Smoking and hospital antenatal services, collected data and supervised others in this. TC wrote the paper and all authors commented on drafts and approved final text. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC529272.xml
555756	Gluten-free diet may alleviate depressive and behavioural symptoms in adolescents with coeliac disease: a prospective follow-up case-series study	Background Coeliac disease in adolescents has been associated with an increased prevalence of depressive and disruptive behavioural disorders, particularly in the phase before diet treatment. We studied the possible effects of a gluten-free diet on psychiatric symptoms, on hormonal status (prolactin, thyroidal function) and on large neutral amino acid serum concentrations in adolescents with coeliac disease commencing a gluten-free diet. Methods Nine adolescents with celiac disease, aged 12 to 16 years, were assessed using the semi-structured K-SADS-Present and Lifetime Diagnostic interview and several symptom scales. Seven of them were followed at 1 to 2, 3, and 6 months on a gluten-free diet. Results Adolescent coeliac disease patients with depression had significantly lower pre-diet tryptophan/ competing amino-acid (CAA) ratios and free tryptophan concentrations, and significantly higher biopsy morning prolactin levels compared to those without depression. A significant decrease in psychiatric symptoms was found at 3 months on a gluten-free diet compared to patients' baseline condition, coinciding with significantly decreased coeliac disease activity and prolactin levels and with a significant increase in serum concentrations of CAAs. Conclusion Although our results of the amino acid analysis and prolactin levels in adolescents are only preliminary, they give support to previous findings on patients with coeliac disease, suggesting that serotonergic dysfunction due to impaired availability of tryptophan may play a role in vulnerability to depressive and behavioural disorders also among adolescents with untreated coeliac disease.	Background Coeliac disease is an under-diagnosed autoimmune type of gastrointestinal disorder resulting from gluten ingestion in genetically susceptible individuals. Non-specific symptoms such as fatigue and dyspepsia are common, but the disease may also be clinically silent. Diagnosis is based on small-bowel biopsy, and a permanent gluten-free diet is the essential treatment. Undetected or neglected, coeliac disease is associated with serious complications. [ 1 - 3 ] Depressive symptoms [ 4 , 5 ] and disorders [ 6 ] are common among adult patients with coeliac disease, and depressive and disruptive behavioural disorders are highly common also among adolescents, particularly in the phase before diet treatment [ 7 ]. Recently 73% of patients with untreated coeliac disease – but only 7% of patients adhering to a gluten-free diet – were reported to have cerebral blood flow abnormalities similar to those among patients with depressive disorders [ 8 ]. Improvement in state anxiety [ 5 ], in behavioural symptoms [ 9 ], and in depressive disorders [ 6 , 10 ] may occur after the start of a standard gluten-free diet, and after a vitamin B-6-supplemented gluten-free diet [ 11 ]. In some cases, however, the more serious depressive episodes have appeared following the commencement of a gluten-free diet [ 6 ]. Mechanisms involved have remained unclear. Some studies have suggested the possibility of impaired availability of tryptophan and disturbances in central serotonergic function as playing a role [ 9 , 12 ]. In parallel with this, a significant increase in major serotonin and dopamine metabolite concentrations in the brain has been reported after one year on a gluten-free diet [ 13 ]. The present work is a preliminary prospective psychiatric follow-up study of adolescents with newly diagnosed coeliac disease measuring psychiatric symptoms, hormonal status (prolactin, thyroidal function), and large neutral amino acid (LNAA) serum concentrations repeatedly after their commencement of a gluten-free diet, testing the hypothesis that the treatment of coeliac disease may increase the availability of tryptophan and alleviate psychiatric symptoms. Methods Subjects The study sample comprised all nine adolescents (5 girls, 4 boys; aged 14.6 ± 0.8) consecutively diagnosed with coeliac disease between January 1999 and December 2000 in the Department of the Gastrointestinal Services of the Hospital for Children and Adolescents, Helsinki University Central Hospital, in Finland. None of the patients had a history of, or current psychiatric treatment. Duration of coeliac disease symptoms and signs (abdominal pain, diarrhoea, anaemia) leading to a biopsy was 2.3 (± 1.5) years. The study was approved by the institutional Ethics Committee. Written informed consent was obtained from each patient and a parent. Evaluation Baseline psychiatric evaluation was conducted 1 to 4 weeks after the diagnostic biopsy, during the wait for the diagnosis of coeliac disease to be established by the pathologist. The adolescent and a parent were interviewed separately by an adolescent psychiatrist (PP) using a semi-structured diagnostic interview, the Schedule for Affective Disorders and Schizophrenia for School-Age Children – Present and Lifetime version (K-SADS-PL) [ 14 ]. Seven patients attended the follow-up visits with laboratory tests at > 1 to ≤ 2, 3, and 6 months after starting a gluten-free diet (Table 1 ). Baseline and follow-up behavioural problems were assessed with the Youth Self Report (YSR) [ 15 ] and the Child Behavior Checklist (CBCL) [ 16 ], completed by a parent (Table 1 ), and depressive and anxiety symptoms by the 21-item versions of the Beck Depression Inventory (BDI) and Beck Anxiety Inventory (BAI), the 17-item version of the Hamilton Depression Rating Scale (HAM-D), and the 14-item Hamilton Anxiety Rating Scale (HAM-A). CGAS (Children's Global Assessment) served as a part of the K-SADS-PL. Table 1 Psychiatric symptoms and disease activity among adolescent CD patients (n = 7, mean ± SD) 0 (baseline) 1–2 months p-value 1) 3 months p-value 1) 6 months p-value 2) CGAS 3) 74.4 (± 16.5) 4) 86.9 (± 5.6) 0.043 88.3 (± 9.2) 0.006 HAM-D 5) 5.7 (± 7.3) 0.3 (± 0.8) 0.043 1.0 (± 1.3) 0.009 HAM-A 6) 6.9 (± 6.9) 0.1 (± 0.4) 0.043 0.7 (± 1.3) 0.010 BDI 7) 3.4 (± 6.5) 0.0 (± 0.0) 8) n.s. 9) 0.1 (± 0.4) 0.041 0.6 (± 1.1) 0.014 BAI 10) 3.0 (± 2.9) 1.3 (± 1.5) 8) n.s. 0.7 (± 1.1) 0.042 1.4 (± 2.2) n.s. CBCL 11) Total problems 28.3 (± 12.8) 18.4 (± 6.9) 0.028 14.0 (± 9.5) 0.028 18.4 (± 16.2) 0.007 Anxious/depressed 3.9 (± 3.1) 1.4 (± 1.7) 0.046 1.3 (± 1.5) 0.026 1.3 (± 1.1) 0.033 Aggressive behaviour 7.3 (± 4.2) 5.6 (± 2.2) n.s. 3.6 (± 3.1) 0.039 6.0 (± 6.4) 0.047 YSR 12) Total problems 24.4 (± 13.8) 17.0 (± 12.5) n.s. 8.0 (± 7.8) 0.018 9.4 (± 7.4) 0.001 Anxious/depressed 2.7 (± 3.4) 1.6 (± 2.2) n.s. 0.3 (± 0.5) 0.026 0.6 (± 1.0) 0.002 Aggressive behaviour 5.1 (± 2.7) 4.1 (± 2.7) n.s. 2.0 (± 2.2) 0.044 2.4 (± 1.4) 0.021 Somatic complains 4.9 (± 2.4) 3.1 (± 2.3) 0.038 2.1 (± 1.2) 0.026 2.3 (± 2.1) 0.003 S-EndoAbA 8) 800(200–1600) 200 (5–400) 0.018 5 (5–1600) 0.027 5 (5–100) 0.001 S-tTGAbA 139 (± 158) 30 (± 35) 0.018 19 (± 26) 0.018 7 (± 8) < 0.001 Prolactin (mU/l) 13) 1569 (± 767) 218 (± 60) 0.028 284 (± 170) 0.018 205 (± 92) 14) 0.019 S-T4/TSH (nmol/l:mU/l) 34 (± 6) 64 (± 38) 14) 0.043 84 (± 53) 0.043 74 (± 32) 15) 0.039 1) non-parametric Wilcoxon signed-ranks test; compared with baseline; 2) repeated measures non-parametric Friedman test; 3) Children's Global Assessment; 4) mean ± SD; 5) Hamilton Depression Rating Scale; 6) Hamilton Anxiety Rating Scale ; 7) Beck Depression Inventory; 8) median (min-max); 9) non significant; 10) Beck Anxiety Inventory; 11) Child Behavior Checklist; 12) Youth Self Report; 13) normal: females 50–300, males 50–500; 14) n = 6 ; 15) n = 4 Coeliac disease activity was followed by determining serum tissue transglutaminase (S-tTGAbA) and endomysium (S-EndoAbA) autoantibodies [ 17 ]. Pre-diet blood samples for analysis of amino acids, prolactin, thyroid function [thyroxine (S-T4), thyroid-stimulating hormone (TSH)], vitamins B6 and B12, S-tTGAbA, and S-EndoAbA were obtained on the biopsy morning, and subsequent ones as a part of follow-up visits, both after overnight fasting, between 8 and 10 a.m. All nine patients had amino acid concentrations measured at baseline, and five of them during follow-up (1–2 times). A blood sample (2 ml) was drawn from the ulnar vein into a vacuum tube for serum total and free L-tryptophan, and for other large neutral amino acids (LNAA). The tube was cooled immediately and stored refrigerated in ice until centrifuged. After centrifugation, the serum was frozen and stored at -20°C for 4 to 14 months (median 7.5) until its assay for the amino acids by a modified procedure described by Qureshi et al. [ 18 ]. All the samples were analysed in a single run, in Kuopio, Finland, and free and total L-tryptophan and other LNAA's were assessed as described by Tiihonen et al. [ 19 ]. Statistical methods Statistical analysis was carried out with parametric and non-parametric tests as appropriate; tests for two-independent groups (T-test, Mann-Whitney U-test), for repeated measures of two-related groups (Wilcoxon signed-ranks test), for three-related groups (Friedman test) and Spearman's rank correlation testing were used. P-values (2-tailed) < 0.05 were regarded as significant. Results Baseline evaluation At baseline, three adolescents (3/9; 33%) had a depressive disorder: two girls had major depressive disorder (MDD), one with a learning disorder not otherwise specified (NOS), and another with comorbid conduct disorder; one girl had the depressive disorder NOS. Further, one boy had a phobic disorder plus attention-deficit hyperactivity disorder, and another conduct disorder NOS. Four adolescents (44%) had no diagnosis. Pre-diet free L-tryptophan was positively correlated with pre-diet levels of tTGAbA (n = 8; r = 0.78, P= 0.022), and negatively with vitamin B-6 (r = 0.73, P = 0.039) and S-T4 (r = 0.74, P = 0.035). Prolactin levels (Table 1 ) from the biopsy morning showed a positive correlation with BDI score (self-report depression inventory; r = 0.89, P = 0.001), and a negative correlation with the ratio of L-tryptophan to amino acids competing for the same cerebral uptake mechanism (CAA) (r = 0.68, P = 0.042), but not with free L-tryptophan levels. The sum of branched-chain amino acids (BCAA: valine, leucine, and isoleucine) showed no correlation with L-tryptophan or free L-tryptophan levels. Depressive patients (n = 3/9) had significantly higher pre-diet prolactin levels (mU/l: mean ± S.D. = 2450 ± 676 vs. 1194 ± 598, Mann-Whitney U-test, P = 0.039) and pre-diet S-T4 levels (nmol/l: mean 102 ± 3.1 vs. 84 ± 16.1, Mann-Whitney U-test, P = 0.024), and significantly lower L-tryptophan/CAA ratios (100 × pmol/μl: pmol/μl: mean 10.0 ± 0.2 vs. 11.5 ± 1.7, Mann-Whitney U-test, P = 0.020) and free L-tryptophan concentrations (pmol/μl: mean 4.7 ± 0.5 vs. 8.4 ± 3.0, two-independent samples T-test, P = 0.029). Pre-diet free L-tryptophan correlated negatively with biopsy morning S-T4 level (r = -0.74, P = 0.035). No significant differences appeared in L-tryptophan (36.3 ± 5.1 vs. 43.3 ± 6.1) or in L-tyrosine concentrations, nor in BCAA and CAA levels. Follow-up Two adolescents with conduct disorders, one a girl with concomitant MDD, did not adhere to the gluten-free diet and dropped out of the psychiatric follow-up. Among others (n = 7), a significant decrease in most of the problem and symptom scores of YSR and CBCL, and in BDI, BAI, and Hamilton scales was evident after 3 months on the gluten-free diet, compared to baseline (Table 1 ). Celiac disease-associated antibody titres had decreased in all by the first month on a gluten-free diet, and had already normalised (= S-EndoAbA titre < 5 and S-tTGAbA titre < 8) in 4 of 7 patients by 6 months. Boys had lower biopsy morning prolactin levels (mU/l; mean 972, SD 450 vs. girls mean 2126 ± 756; one-way Anova P = 0.032), but higher levels after one month on the diet. In the first month, the S-T4/TSH ratio (nmol/l:mU/l) reflecting thyroid function increased significantly (Table 1 ). An initial increase in CAAs, also in tyrosine levels, and in total and free L-tryptophan was reaching significance after one month on a gluten-free diet. By 3 months, the increases in tyrosine alone and in CAAs as a group were significant, and the increase in free L-tryptophan was approaching significance (repeated measures Friedman test n = 4, Chi-Square 6,000, df 2, P = 0.050). (Table 2 ) Table 2 Follow-up of the patients (n = 5): psychiatric symptoms, CD activity, and amino acidconcentrations [median (min-max)]. 0 (baseline) ≥1<1.5 months 1) p-value 2) ≥3 months p-value 2) CBCL Total problems 34 (9–39) 21 (8–29) 21 (2–22) YSR Total problems 20 (9–36) 11 (5–41) n.s. 2 (0–22) S-tTGAbA 42 (10–310) 5 (3–51) 3 (1–17) ** Prolactin 3) 1100 (635–2850) 256 (127–282) n.s * 244 (110–565) ** L-tyrosine 4) 33 (26–44) 39 (37–70) n.s * 40 (38–47) ** CAA 5) 353 (316–441) 401 (368–657) n.s * 395 (367–568) ** L-tryptophan 4) 40 (32–51) 52 (45–66) n.s * 46 (36–59) n.s. Tryptophan/CAA 6) 11.3 (10.1–14.9) 12.0 (10.1–14.1) n.s. 10.1 (9.5–12.5) n.s. Free L-tryptophan 4) 4.9 (4.5–11.8) 8.4 (5.3–10.9) n.s * 10.6 (5.0–19.0) n.s. * 7) Free tryptophan/CAA 6) 1.4 (1.0–3.5) 1.9 (1.0–3.0) n.s. 2.6 (1.3–3.4) n.s. 1) amino acid concentrations; n = 4; 2) Wilcoxon signed – ranks test: compared with baseline; ** = P < 0.05; * = P ≥ 0.05 < 0.07; 3) mU/l; normal: females 50–300, males 50–500; 4) pmol/μl; 5) L-valine, L-leucine, L-isoleucine, L-phenylalanine, L-tyrosine; pmol/μl; 6) 100 × pmol/μl: pmol/μl; 7) repeated Measures Friedman test, n = 4, P = 0.050 Discussion We observed that the majority of adolescents with coeliac disease had depressive and behavioural symptoms before their diagnosis, and that coeliac disease patients with depression (all girls) had significantly lower pre-diet tryptophan/CAA ratios and free tryptophan concentrations and significantly higher biopsy morning prolactin levels. Adolescents with coeliac disease showed improvement in psychiatric symptoms after starting a gluten-free diet, and this improvement coincided with a significant decrease in coeliac disease activity and in prolactin levels, and with a significant increase in serum concentrations of L-tyrosine and other CAAs. The increase in free L-tryptophan levels was approaching significance. The findings of this study – improvement in depressive and behavioural symptoms after the start of a gluten-free diet – are supported by the findings of our larger previous retrospective case-control study [ 7 ]. Although the results of the amino acid analysis and prolactin levels are only preliminary, they give support to the hypothesis that impaired availability of tryptophan and the possible consequent serotonergic dysfunction may play a role in vulnerability to depressive disorders among adolescents with untreated coeliac disease. A possible role for tyrosine and the brain's catecholamine metabolism (dopamine and noradrenaline) in these disorders cannot, however, be excluded. The decrease observed in psychiatric symptoms took place regardless of the stress accompanying being diagnosed with a chronic and restrictive illness, and improvement was not explainable in terms of physical symptoms, since both in the present and in our previous study [ 7 ], the presence or alleviation of depression showed no association with somatic symptom severity. Our results from adolescents differ from those reported by Addolorato et al. [ 5 ]. In their follow-up study on adult patients with coeliac disease, a significant decrease in anxiety symptoms but not in depressive symptoms appeared after one year on a gluten-free diet. Although converging with the findings of Ljungman and Myrdal (20), the few symptoms of our adolescents with coeliac disease adhering to a gluten-free diet in our present and previous [ 7 ] studies are thus in contrast to the findings of depressive symptoms [ 4 , 5 ] and disorders [ 6 ] as being common among adult patients with coeliac disease, even during diet treatment. Since the free tryptophan and the tryptophan/CAA ratios in plasma determine the availability of tryptophan to the brain [ 21 ], our findings on depressive patients give preliminary support to suggestions of impaired availability of tryptophan as featuring in coeliac-associated depressive and behavioural disorders associated with celiac disease [ 9 , 12 , 13 ]. As we did not have a control group of healthy adolescents, we cannot say whether L-tryptophan or L-tyrosine levels or both are generally lower among adolescents with coeliac disease, as could be expected based on the findings of Hernanz and Polanco [ 9 ], who reported significantly decreased plasma tryptophan and tyrosine concentrations in untreated and treated children with coeliac disease compared to levels in controls. In the present study, stress-induced biopsy-morning prolactin levels were significantly higher among depressive patients (all girls) and correlated negatively with L-tryptophan/CAA levels. This finding is only preliminary, but it is, however, interesting: Although disturbances in the central serotonergic system have been associated with depressive and impulse-control disorders among adults [see [ 22 ]], and children aged 6 to 12 years with a recent suicide attempt have shown lower whole blood tryptophan content [ 23 ], serotonergic dysfunction in adolescents with depression is still poorly studied. The prolactin hypersecretion response to the L-5-hydroxytryptophan challenge (L-5HTP) test reported among pre-pubertal girls with major depressive disorder [ 24 ] and among healthy children at high risk for major depressive disorder (= high family loading for major depression) [ 25 ], may be consistent with dysregulation of the central serotonergic system in childhood major depression [ 24 ]. Moreover, alterations in neuroendocrine responses to L-5HTP challenge tests, such as the prolactin hypersecretion and hyposecretion of cortisol found in healthy children, have been suggested to represent a trait marker for depression in children [ 25 ]. Thus, the high biopsy morning prolactin levels in depressed adolescents with untreated coeliac disease in the present study could be associated with serotonergic dysfunction. They could also be associated with dopaminergic dysfunction due to impaired availability of tyrosine, since dopamine is known to exert an inhibitory action on prolactin release in the hypothalamus [ 26 ]. In the present study, however, pre-diet prolactin levels did not correlate with tyrosine levels. Moreover, the function of the intestinal Catechol-O-Methyl Transferase enzyme (COMT) – known to play an important role in the peripheral O-methylation of catecholamines – remains unstudied in untreated coeliac disease. It is of some theoretical interest that reduced COMT activity in erythrocytes has at least once been associated with conditions such as primary affective disorders in women [ 27 ]. On the other hand, in the present study also non-depressed adolescents with coeliac disease had higher than normal biopsy morning prolactin levels. Significantly higher prolactin levels among untreated coeliac children (5–18 years) compared with treated patients has been reported by Reifen et al. [ 28 ]. They suggest that prolactin may play a part in the immune modulation of the intestine and could thus serve as a potential marker for coeliac disease activity. Our preliminary findings on amino acid levels in adolescents with coeliac disease with or without depression are unlikely to be explained by malabsorption, since pre-diet free L-tryptophan and tryptophan ratios were not correlated with the BCAA levels that reflect the level of protein nutrition. It is of theoretical interest that increased production of interferon-γ (IFN-γ), known to be the predominant cytokine produced by gluten-specific T-cells in active coeliac disease [ 29 ], can suppress serotonin function both directly and indirectly by enhancing tryptophan and serotonin turnover [ 30 ]. Increased IFN-γ [ 30 ] and, for instance, such events as a stress-related increase in liver tryptophan pyrrolase enzyme activity [ 23 ], may lead to lowered tryptophan levels by the enhanced tryptophan catabolism induced by increased activity of the kynurenine-niacin pathway [ 30 - 32 ], even without malabsorption. Conclusion The alleviation of psychiatric symptoms found among adolescents with coeliac disease after commencement of a gluten-free diet coincides with a rapid decrease in antibody titres indicating coeliac disease activity and in their prolactin levels, and with a significant increase in L-tyrosine and other CAA serum concentrations, and with a nearly significant increase in the free fraction of L-tryptophan. Although these findings are only preliminary, and more research is needed, they give support to previous findings on patients with coeliac disease, suggesting that serotonergic dysfunction due to impaired availability of tryptophan may play a role in vulnerability to depressive and behavioural disorders, also among adolescents with untreated celiac disease. And since diet treatment may alleviate psychiatric symptoms, and earlier diagnosis may have beneficial effects on psychological and even on neurobiological vulnerability to depression, the possibility of psychiatric complications of coeliac disease needs to be taken into account in differential diagnosis of depressive and behavioural disorders. Declaration of competing interests The author(s) declare that they have no competing interests. Authors' contributions PAP, ETI, MAV, ES, VAA contributed to the conception and design of the study, and PAP, MAV, ES to acquisition of the data. All authors (PAP, ETI, MAV, SAK, IS, ES, VAA) contributed to the analysis and interpretation of the data, were involved in drafting and revising the article, and read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555756.xml
555742	HLA class I and II genotype of the NCI-60 cell lines	Sixty cancer cell lines have been extensively characterized and used by the National Cancer Institute's Developmental Therapeutics Program (NCI-60) since the early 90's as screening tools for anti-cancer drug development. An extensive database has been accumulated that could be used to select individual cells lines for specific experimental designs based on their global genetic and biological profile. However, information on the human leukocyte antigen (HLA) genotype of these cell lines is scant and mostly antiquated since it was derived from serological typing. We, therefore, re-typed the NCI-60 panel of cell lines by high-resolution sequence-based typing. This information may be used to: 1) identify and verify the identity of the same cell lines at various institutions; 2) check for possible contaminant cell lines in culture; 3) adopt individual cell lines for experiments in which knowledge of HLA molecule expression is relevant. Since genome-based typing does not guarantee actual surface protein expression, further characterization of relevant cell lines should be entertained to verify surface expression in experiments requiring correct antigen presentation.	Background A panel of sixty cancer cell lines of diverse lineage (lung, renal, colorectal, ovarian, breast, prostate, central nervous system, melanoma and hematological malignancies) was developed, characterized and extensively used by the National Cancer Institute's Developmental Therapeutics Program (NCI-60) since the early 90's as a screening tool for anti-cancer drug development [ 1 ]. This strategy [ 2 - 9 ]. yielded data about drug-related cytotoxicity for about 100,000 compounds. In addition, extensive functional characterization of the NCI-60 response to diverse biological or chemical stimulation has been accumulated [ 10 - 15 ]. Although originally developed for chemo-sensitivity testing, with the development of high-throughput analyses the NCI-60 panel has been broadly characterized for other biological applications [ 16 - 25 ]. Thus, patterns incidentally identified provided platforms for further investigations of mechanisms of tumorigenesis and cancer progression [ 5 , 6 , 26 - 30 ]. More recently, genomic DNA [ 24 ] and proteomics analyses have further characterized the profile of these cell lines [ 31 ]. The combined database provides the most comprehensive phenotyping of commonly accessible cancer cell lines offering correlative information about genetic, transcriptional and post-translational qualities. With growing interest in the identification of novel tumor antigens recognized by T cells as targets for antigen-specific immunization ([ 32 ], the NCI-60 could become an ideal tool for in silico discovery [ 33 ] ([ 34 ] and for tumor cell-specific T-cell reactivity testing [ 35 ]. For this purpose, accurate information about the extended human leukocyte antigen (HLA) phenotype of each cell line is necessary for the definition and validation of specific HLA/epitope combinations. Although antiquated and partial information about the HLA phenotype of some of the NCI-60 cell lines is available through the American Type Culture Collection (ATCC), Rockville, MD, no high-resolution information obtained by definitive sequence-based typing (SBT) has ever been published. Since T cell recognition of HLA-epitope complexes is narrowly restricted to unique combinations [ 36 ], this information is critical to select reasonable candidates for antigen-discovery choosing cell lines bearing HLA phenotypes most relevant to the disease population studied [ 37 ]. Accurate information about the HLA genotype of each cell line may, in addition, help their identification, validation and qualification among different laboratories excluding possible errors related to switching of cell lines or culture contamination. Therefore, we provide high-resolution SBT of the complete NCI-60 panel obtained from their original source: the National Cancer Institute's Developmental Therapeutics Program. Results and Discussion Previous knowledge of the HLA phenotype of NCI-60 cell lines We reviewed and collected available information about the HLA phenotype of the NCI-60 cell lines, performed according to serological testing before submission to the ATCC (Table 1 ). The information was collected through the ATCC website: . Most cell lines had not been previously typed; the large majority of the cell lines from which such information is available had been developed from Caucasian patients. HLA typing was reported according to the old serologic nomenclature at a very low level of resolution. In addition, several reported typings did not match the present typing as shown in Table 2 and 3 . This was the case for the colon carcinoma cell line HT29 that maintained a correct haplotype (with the exclusion of the HLA-Cw locus) but had a completely different second haplotype. The melanoma cell line SK-MEL-5 had an almost identical haplotype with the exception of one HLA-B allele originally typed as Bw16 (inclusive of the molecularly-defined alleles: B38 and B39), while the present typing was HLA-B07. Another melanoma cell line SK-MEL-28 maintained a haplotype similar to the previously reported HLA-A11, -B40 but appeared to have lost an HLA-A allele (HLA-A26) compared with the original ATCC description. Finally, the multiple myeloma cell line RPMI 8226 was matched at one haplotype (HLA-A19, -B15 and -Cw2) but was totally discrepant at the second haplotype (HLA-A6802, -B1510 and -Cw0304). The HLA typing of the other two previously typed cell lines was confirmed in the present study. Overall, in spite of the discrepancies in HLA typing observed between the previous and the present analyses, a resemblance was noted in the cell line genotype suggesting that mis-typing related to the low accuracy of serological methods might have been at the basis of the discrepancy rather than contamination or switching of the cell lines. Table 1 Available information from the ATCC about the NCI-60 panel Name ATCC no. Sex Race Tumor Type ATCC HLA typing Discrepant BT-549 HTB-122 F C Breast CA HS 578T HTB-126 F C Breast CA MCF7 HTB-22 F C Breast CA MDA-MB-231 HTB-26 F C Breast CA MDA-MB-435 HTB-129 F C Breast CA T-47D HTB-133 F Breast CA SF-268 CNS CA SF-295 CNS CA SF-539 CNS CA SNB-19 CNS CA SNB-75 CNS CA U251 CNS CA COLO 205 CCL-222 M C Colon CA HCC-2998 Colon CA HCT-116 CCL-247 M Colon CA HCT-15 CCL-225 M Colon CA HT29 HTB-38 F C Colon CA A1,3,B12,17 Cw5 Yes KM12 Colon CA SW-620 CCL-227 M Colon CA MOLT-4 CRL-1582 M Leukemia, ALL CCRF-CEM CCL-119 F C Leukemia, ALL HL-60 CCL-240 F C Leukemia, APL K-562 CCL-243 F Leukemia, CML SR CRL-2262 M C Leukemia, LCIL LOX IMVI Melanoma M 14 Melanoma SK-MEL-2 HTB-68 M C Melanoma SK-MEL-5 HTB-70 F C Melanoma A2,11, B40,Bw16 Yes SK-MEL-28 HTB-72 M Melanoma A11,26, B40,DRw4 Yes UACC-62 Melanoma UACC-257 Melanoma RPMI 8226 CCL-155 M MM Aw19, B15,37, Cw2 Yes A549/ATCC CCL-185 M C NSCLC EKVX NSCLC HOP-62 NSCLC HOP-92 NSCLC NCI-H23 CRL-5800 M AA NSCLC NCI-H226 CRL-5826 M NSCLC NCI-H322M NSCLC NCI-H460 HTB-177 M NSCLC NCI-H522 CRL-5810 M C NSCLC IGROV1 Ovarian CA OVCAR-3 HTB-161 Ovarian CA OVCAR-4 Ovarian CA OVCAR-5 Ovarian CA OVCAR-8 Ovarian CA NCI/ADR-RES Ovarian CA SK-OV-3 HTB-77 F C Ovarian CA DU-145 HTB-81 M C Prostate CA PC-3 CRL-1435 M C Prostate CA A1,9 No 786-O Renal CA A498 HTB-44 F Renal CA ACHN CRL-1611 M C Renal CA CAK1-1 HTB-46 M C Renal CA A9,B12,35 No SN12C Renal CA TK-10 Renal CA UO-31 Renal CA RXF-393 Renal CA AA = African American; ALL = Acute Lymphoblastic Leukemia; APL = Acute promyelocytic leukemia; C = Caucasian; CA = Carcinoma; CML = Chronic Myelogenous Leukemia; CNS = Central Nervous System; F = Female; LCIL = Large Cell Immunoblastic Lymphoma; M = Male; MM = Multiple Myeloma; NA = Not Available; NSCLC = Non Small Cell Lung Cancer. The information about the ATCC cell lines (Cell Lines with ATCC no.) was obtained accessing the following URL: . Additional information was obtained through the National Cancer Institute's Developmental Therapeutics Program URL: . Table 2 Sequence-based typing of NCI-60 HLA class I Loci Cell Line ID Tissue A locus B Locus Cw Locus BT-549 41292-D Breast CA N.R. 151701, 5501 030301, 07 a HS 578T 41293-D Breast CA 03 a , 24 a 35 a , 40 a 030401, 04 a MCF7 41294-D Breast CA 020101 18 a , 44 a 05 a MDA-MB-231 41296-D Breast CA 0201, 0217 4002, 4101 020202, 17 a MDA-MB435 41297-D Breast CA 110101, 240201 15 a , 35 a 030301, 04 a T47D 41298-D Breast CA 3301 1402 0802 SF-268 41286-D CNS CA 010101, 3201 0801, 4002 020202, 07 a SF-295 41287-D CNS CA 010101, 2601 070201, 5501 03 a , 07 a SF-539 41288-D CNS CA 020101 08 a , 35 a 04 a , 07 a SNB-19 41289-D CNS CA 020101 18 a 05 a SNB-75 41290-D CNS CA 020101, 110101 35 a , 39 a 04 a , 120301 U251 41291-D CNS CA 020101 18 a 05 a COLO 205 41299-D Colon CA 01 a , 02 a 07 a , 08 a 070201, 07 a HCC-2998 41300-D Colon CA 02 a , 24 a 3701, 400601 04 a , 0602 HCT-116 41301-D Colon CA 01 a , 02 a 18 a , 4501 05 a , 07 a HCT-15 41302-D Colon CA 02 a , 24 a 08new, 350101 04 a , 07 a HT29 41303-D Colon CA 01 a , 24 a 35 a , 440301 04 a KM12 41304-D Colon CA 02new 70201 70201 SW-620 41305-D Colon CA 02 a , 24 a 07 a , 15 a 070201, 07 a MOLT 4 41281-D Leukemia, ALL 010101, 2501 18 a , 570101 0602, 120301 CCRF-CEM 41282-D Leukemia, ALL N.R. 08 a , 40 a 030401, 07 a HL-60 41284-D Leukemia, APL 10101 570101 0602 K-562 41280-D Leukemia, CML 110101, 310102 18 a , 40 a 03 a , N.R. SR 41285-D Leukemia, LCIL 02 a , 03 a 3701, 3901 0602, 120301 LOX IMVI 41315-D Melanoma 110101, 2902 070201, 440301 070201, 1601 M 14 41316-D Melanoma 110101, 240201 15 a , 35 a 030301, 04 a SK-MEL-2 41317-D Melanoma 03 a , 26 a 35 a , 38 a 04 a , 120301 SK-MEL-5 41319-D Melanoma 020101, 110101 07 a , 40 a 030401, 070201 SK-MEL-28 41318-D Melanoma 110101 4001 030401 UACC-62 41321-D Melanoma 02 a , 32 a 39 a , 44 a 05 a , 12 a UACC-257 41320-D Melanoma 020101 18 a , 44 a 05 a , 07 a RPMI-8226 41283-D MM 3001, 6802 1503, 1510 020204, 030402 A549/ATCC 41306-D NSCLC 2501, 3001 18 a , 440301 120301, 1601 EKVX 41307-D NSCLC 010101 3701 0602 HOP-62 41308-D NSCLC 030101 07 a , 44 a 05 a , 070201 HOP-92 41309-D NSCLC 03 a , 24 a 27 a , 470101 01 a , 06 a NCI-H23 41312-D NSCLC 8001 5001 0602 NCI-H226 41311-D NSCLC 010101, 240201 07 a , 39 a 070201, 120301 NCI-H322M 41310-D NSCLC 2902 440301 1601 NCI-H460 41313-D NSCLC 24 a , 68 a 35 a , 51 a 03 a , 15 a NCI-H522 41314-D NSCLC 020101 44 a , 5501 030301, 05 a IGROV1 41322-D Ovarian CA 240201, 3301 4901 07 a OVCAR-3 41323-D Ovarian CA 020101, 2902 070201, 5801 070201, 07 a OVCAR-4 41324-D Ovarian CA 010101, 3201 0801, 4002 07 a , 15 a OVCAR-5 41325-D Ovarian CA 01 a , 02 a 08 a , 44 a 05 a , 07 a OVCAR-8 41326-D Ovarian CA 010101, 2501 570101 0602 NCI/ADR-RES 41295-D Ovarian CA 010101, 2501 570101 0602 SK-OV-3 41327-D Ovarian CA 03 a , 68 a 18 a , 35 a 04 a , 05 a DU-145 41328-D Prostate CA 030101, 3303 5001, 570101 0602 PC-3 41329-D Prostate CA 010101, 240201 1302, 5501 01 a , 06 a 786-O 41330-D Renal CA 030101 07 a , 44 a 05 a , 070201 A498 41331-D Renal CA 020101 0801 07 a ACHN 41332-D Renal CA 2601 4901 07 a CAKI-1 41333-D Renal CA 2301, 240201 3502, 440301 04 a , 04new SN12C 41334-D Renal CA 03, 24new 07 a , 44 a 05 a , 070201 TK-10 41335-D Renal CA 3301 1402 0802 UO-31 41336-D Renal CA 010101, 030101 07 a , 14 a 07 a , 08 a RXF-393 41337-D Renal CA 02 a , 24 a 1401, 44 a 05 a , 0802 Sequence-based typing for the HLA class I loci are reported with the highest degree of resolution. Non-resolved ambiguities are reported as two digit denominations with a superscript a as previously described 43. HLA typings divergent from those originally described in the ATCC database are reported in red. ID# refers to the HLA laboratory reference number. New alleles are indicated by the suffix new following the allele. N.R. – Ambiguity not resolved at the lower level of resolution. Table 3 Sequence-based typing of NCI-60 HLA class II Loci Cell Line ID Tissue DRβ1 Locus DQB1 Locus DPB1 Locus BT-549 41292-D Breast CA 11 a , 13 a 030101, 060401 020102, 0401 HS 578T 41293-D Breast CA 01 a , 150101 050101, 0602 0401, 7801 MCF7 41294-D Breast CA 03 a , 15 a 0201, 0602 020102, 0401 MDA-MB-231 41296-D Breast CA 0701, 1305 0202, 030101 020102, 1701 MDA-MB435 41297-D Breast CA 040501, 130101 0302, 0603 1301, 1901 T47D 41298-D Breast CA 010201 050101 020102, 0401 SF-268 41286-D CNS CA 03 a , 04 a 0201, 0302 0401, 0601 SF-295 41287-D CNS CA 14 a , 15 a 050301, 0602 0401 SF-539 41288-D CNS CA 030101, 12 a 0201, 030101 010101, 0401 SNB-19 41289-D CNS CA 030101 0201 0402 SNB-75 41290-D CNS CA 0103, 11 a 03 a , 050101 0401, 0402 U251 41291-D CNS CA 030101 0201 0402 COLO 205 41299-D Colon CA 040101, 130101 0603 0401 HCC-2998 41300-D Colon CA 11 a , 16 a 030101, 050201 0401 HCT-116 41301-D Colon CA N.R. 02new, 03new 030101, 0402 HCT-15 41302-D Colon CA 03 a , 14 a 02 a , 050301 010101, 0401 HT29 41303-D Colon CA 0402, 0701 02 a , 0302 0401 KM12 41304-D Colon CA 040101 0302 1301 SW-620 41305-D Colon CA 0103, 130101 050101, 0603 010101, 0401 MOLT 4 41281-D Leukemia, ALL 07new, 12new 0202, 030101 20102 CCRF-CEM 41282-D Leukemia, ALL 030101, 0701 0201, 0202 0401, 1301 HL-60 41284-D Leukemia, APL N.R. 030302 0401, 1301 K-562 41280-D Leukemia, CML 03 a , 04 a 0201, 0302 0401, 0402 SR 41285-D Leukemia, LCIL 01 a , 160101 050101, 050201 0401 LOX IMVI 41315-D Melanoma 0701, 150101 0202, 0602 0401, 110101 M 14 41316-D Melanoma 040501, 130101 0302, 0603 1301, 1901 SK-MEL-2 41317-D Melanoma 0402, 130101 030101, 0603 020102, 0401 SK-MEL-5 41319-D Melanoma 040101, 130101 0302, 0603 030101, 1601 SK-MEL-28 41318-D Melanoma 0404 0302 030101 UACC-62 41321-D Melanoma 12 a , 130101 030101, 0603 0401, 1401 UACC-257 41320-D Melanoma 040101 030101, 0302 0401 RPMI-8226 41283-D MM 030101, 0701 0201, 0202 010102, 1301 A549/ATCC 41306-D NSCLC 0701, 110401 0202, 030101 N.R. EKVX 41307-D NSCLC 150101 0602 0401 HOP-62 41308-D NSCLC 13 a , 15 a 06 a , 06 a 0402 HOP-92 41309-D NSCLC 01 a , 150101 050101, 0602 0401, 0402 NCI-H23 41312-D NSCLC 130101 0603 1901 NCI-H226 41311-D NSCLC 150101, 160101 050201, 0602 020102, 0401 NCI-H322M 41310-D NSCLC 0701 0202 0401 NCI-H460 41313-D NSCLC 01 a , 04 a 030101, 050101 N.R. NCI-H522 41314-D NSCLC 040101, 150101 03 a , 0602 0401 IGROV1 41322-D Ovarian CA 11 a , 11 a 03new new, 0501 OVCAR-3 41323-D Ovarian CA 080101, 080401 0402 020102, 0401 OVCAR-4 41324-D Ovarian CA 030101, 040101 0201, 030101 0401, 1301 OVCAR-5 41325-D Ovarian CA 030101, 040101 0201, 030101 0401 OVCAR-8 41326-D Ovarian CA 0701, 150101 030302, 0602 020102, 1301 NCI/ADR-RES 41295-D Ovarian CA 0701, 150101 030302, 0602 020102, 1301 SK-OV-3 41327-D Ovarian CA 01 a , 030101 0201, 050101 020102, 0401 DU-145 41328-D Prostate CA N.R. 030302, 050101 0401 PC-3 41329-D Prostate CA 0701, 130101 0202, 0603 0401 786-O 41330-D Renal CA 13 a , 15 a 06 a , 06 a 0402 A498 41331-D Renal CA 030101 0201 010101 ACHN 41332-D Renal CA 160101 050201 020102 CAKI-1 41333-D Renal CA 0701, 110401 0202, 03 a 020102, 1001 SN12C 41334-D Renal CA 040101, 150101 03 a , 0602 N.R. TK-10 41335-D Renal CA 010201 050101 0402 UO-31 41336-D Renal CA 130201, 150101 0602, 0609 0402, 0501 RXF-393 41337-D Renal CA 110101, 150101 030101, 0602 010101, 0401 Sequence-based typing for the HLA class II loci are reported with the highest degree of resolution. Non-resolved ambiguities are reported as two digit denominations with a superscript a as previously described [43]. HLA typings divergent from those originally described in the ATCC database are reported in red. ID# refers to the HLA laboratory reference number. New alleles are indicated by the suffix new following the allele. N.R. = Ambiguity not resolved at the lower level of resolution. Overall, there was no evidence of contamination among the cell lines tested with clean homozygous or heterozygous combinations observed in all loci analyzed. SBT of HLA class I and HLA class II loci are reported in Table 2 and 3 respectively. Information about the HLA typing of the cell lines is also available through the Molecular Targets URL: . Approximately 17% of the cell lines (10 out of 58 including: T47D, SNB-19, U251, KM12, RPMI-8226, EKVX, NCI-H23, NCI-H322M, A498, ACHN and TK-10) exhibited a pseudo-homozygous pattern suggestive of complete loss of heterozygosity encompassing the HLA class I and HLA class II regions. This frequency is close to the loss of haplotype that we originally described for melanoma cell lines generated at the National Cancer Institute (Bethesda, MD) [ 38 , 39 ] and subsequently observed in other cancers [ 40 , 41 ]. We conclude that this is an unlikely representative of patients' homozygosity because complete HLA class I and II homozygosity is exceedingly rare in the population at large. To corroborate this statement, we analyzed 554 genomic DNA specimens from normal donors recently typed with the same technology in our laboratory. Genomic DNA for the normal donors was obtained from whole blood samples. Only 5 individuals were found to be truly homozygous for all HLA class I and class II loci for a frequency of 0.9%. Overall, discrepancies between ATCC typings and the present typing or the unbalanced frequency of homozygosity could be related to accumulated genetic alterations between the cell lines since the time of their original expansion from the patient and should not be surprising. A particular case was represented by the NCI/ADR-RES cell line which was previously believed to be an adriamycin derivative of the breast cancer cell line MCF-7. Subsequently, it was discovered not to be related to MCF-7, but it's derivation was unclear [ 42 ]. Karyotyping analysis suggested it was related to the ovarian cell line OVCAR-8. Subsequent DNA fingerprinting confirmed that both cell lines were generated from the same individual. HLA genotyping confirms this since the cell lines are indeed identical. To avoid possible misinterpretations, a large number of alleles are not presented here with their definitive nomenclature but rather at a two digits level of resolution because some of the ambiguities could not be completely resolved by SBT as previously described [ 43 ]. However, more detailed information about individual cell lines can be obtained by contacting Sharon Adams directly at the HLA laboratory, Department of Transfusion Medicine, Bethesda, MD. As previously described [ 43 ], it is possible to resolve most of these ambiguities using various methods including sequence-specific primer PCR or pyro-sequencing [ 44 ]. If necessary in the future, the NIH HLA laboratory may assist in further characterization of individual HLA alleles. Another caveat is that the identification of HLA alleles at the genomic level does not necessarily correspond to surface expression of their protein products since various abnormalities in transcription, translation and assembling could influence the surface expression of HLA molecules [ 39 , 45 , 46 ]. Finally, several new alleles were identified (referred to in the tables as new, for which a nomenclature is pending; in detail KM12 HLA-A02new = Genebank Accession # AY918166; SN12C HLA-A24new = # AY918167; CAKI-1 HLA-Cw04new = # AY918170). Information regarding the sequence of these alleles could be obtained by directly contacting the HLA laboratory, Department of Transfusion Medicine, Bethesda, MD. Materials and Methods Cell Lines Genomic DNA from the NCI-60 cell line anticancer drug discovery panel was obtained from SH of the National Cancer Institute Developmental Therapeutics Program (Bethesda, MD). Cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and 5 mM L-glutamine. DNA Isolation Genomic DNA was isolated from peripheral blood using the Gentra PUREGENE isolation kit (Gentra Systems, Minneapolis, MN, USA). The DNA was re-suspended in Tris HCl buffer (pH 8.5) and the concentration was measured using a Pharmacia Gene Quant II Spectrophotometer. The DNA was then stored at -70°C until testing. Sequence-Based Typing (SBT) HLA class I loci sequence-based typing (SBT) was performed as previously described ([ 43 ]. The primary PCR amplification reaction produced a 1.5 kb amplicon encompassing exon 1 through intron 3 of the HLA class I locus. All reagents necessary for primary amplification and sequencing were included in the HLA-A, HLA-B and HLA-C alleleSEQR Sequenced Based Typing Kits (Atria Genetics, Hayward, CA, U.S.A.). The primary amplification PCR products were purified from excess primers, dNTPs and genomic DNA using ExoSAP-IT (American Life Science, Cleveland, OH, U.S.A.). Each template was sequenced in the forward and reverse sequence orientation for exon 2 and exon 3 according to protocols supplied with the SBT kits. Excess dye terminators were removed from the sequencing products utilizing an ethanol precipitation method with absolute ethanol. The reaction products were reconstituted with 15 μl of Hi-Di™ Formamide (PE Applied Biosystems / Perkin-Elmer, Foster City, CA, U.S.A.) and analyzed on the ABI Prism* 3700 DNA Analyzer with Dye Set file: Z and mobility file: DT3700POP6 [ET].	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555742.xml
524030	Diet induced thermogenesis	Objective Daily energy expenditure consists of three components: basal metabolic rate, diet-induced thermogenesis and the energy cost of physical activity. Here, data on diet-induced thermogenesis are reviewed in relation to measuring conditions and characteristics of the diet. Methods Measuring conditions include nutritional status of the subject, physical activity and duration of the observation. Diet characteristics are energy content and macronutrient composition. Results Most studies measure diet-induced thermogenesis as the increase in energy expenditure above basal metabolic rate. Generally, the hierarchy in macronutrient oxidation in the postprandial state is reflected similarly in diet-induced thermogenesis, with the sequence alcohol, protein, carbohydrate, and fat. A mixed diet consumed at energy balance results in a diet induced energy expenditure of 5 to 15 % of daily energy expenditure. Values are higher at a relatively high protein and alcohol consumption and lower at a high fat consumption. Protein induced thermogenesis has an important effect on satiety. In conclusion, the main determinants of diet-induced thermogenesis are the energy content and the protein- and alcohol fraction of the diet. Protein plays a key role in body weight regulation through satiety related to diet-induced thermogenesis.	Introduction Diet induced thermogenesis (DIT) can be defined as the increase in energy expenditure above basal fasting level divided by the energy content of the food ingested and is commonly expressed as a percentage. It is, with basal metabolic rate and activity induced thermogenesis, one of the three components of daily energy expenditure. Although DIT is the smallest component, it could play a role in the development and/or maintenance of obesity. De Jonge and Bray [ 1 ] evaluated 49 studies that compared DIT in subjects who were obese with those who were lean. Of 29 studies, in which the subjects with obesity had a significantly higher body mass index compared with the lean individuals, and the two groups were well matched for age, 22 studies reported a significantly lower DIT in the subjects with obesity. Granata and Brandon [ 2 ] suggested the theory that DIT is reduced in obesity appears to be attractive and plausible, yet discrepant findings persist in the literature and research has uncovered numerous flaws and concerns regarding the methods used to measure and calculate DIT. Methodological issues include: was the baseline appropriate, what was the energy content and nutrient composition of the test food consumed, what was the duration of the postprandial measurement period, and what was the error associated with the calculation of DIT from the measured energy expenditure. Weststrate et al [ 3 ] investigated whether repeated measurements varied with time of day and found no significant diurnal variation in DIT. The postprandial rise in energy expenditure lasts for several hours and is often regarded as completely terminated at approximately 10 hours after the last meal but there is still an argument as to when the post absorptive state is reached. Reed and Hill [ 4 ] analyzed 131 DIT tests from a wide range of subjects ingesting meals of varying sizes and composition. Each test lasted 6 h. They concluded that DIT is a response lasting more than 6 h, especially in obese subjects. Many methodological problems in the measurement of DIT like the choice of meal size and the length of the measurement interval can be circumvented by measuring DIT over 24 h in a respiration chamber [ 5 , 6 ]. Then, activity associated energy expenditure is subtracted from 24 h energy expenditure leaving basal metabolic rate and DIT. Here, the focus is on DIT as a function of the energy content and nutrient composition of the test food consumed and the duration of the postprandial measurement period in adult subjects with a normal bodyweight. The review is based on literature published over the last 15 years. Methods The experimental design of most studies on DIT is a measurement of resting energy expenditure before and after a test meal, with a ventilated hood system. The observation is started after an overnight fast, where subjects are refrained from eating after the last meal at 20.00 h at the latest. Thus, with observations starting between 08.00 and 09.00 h the next morning, the fasting interval is at least 12 h. Postprandial measurements are made for several hours where subjects have to remain stationery, most often in a supine position, for the duration of the measurements. In some studies, measurements are 30 min with 15 min intervals allowing i.e. for sanitary activities. The use of a respiration chamber to measure DIT has the advantage of reproducing more physiological conditions over a longer period of time while regular meals are consumed throughout the day [ 5 , 6 ]. The DIT, as observed in a respiration chamber over 24 h has been evaluated in different ways: 1) as the difference in 24-h energy expenditure between a day in the fed state and a day in the fasted state; 2) as the difference in daytime energy expenditure adjusted for the variability of spontaneous activity and basal metabolic rate; and 3) as the difference in 24-h energy expenditure adjusted for the variability of spontaneous activity and basal metabolic rate. Studies on DIT were selected from Medline. Studies were selected when information was presented on energy intake, diet composition with respect to carbohydrate, protein fat and alcohol of the test food, duration of the postprandial measurement, and DIT. Results Reported intra-individual variability in DIT, determined with ventilated-hood systems, is 6 to 30% [ 7 , 8 ]. Reported within-subject variability in DIT, determined with a respiration chamber, is 43 to 48% [ 5 , 9 ]. The figures for the respiration chamber measurements are for the 24-h DIT calculation as described above under method 3. Method 2, daytime DIT calculation, resulted in an intra-individual variability of 125% [ 5 ]. The mean pattern of DIT throughout the day is presented in figure 1 . Data are from a study where DIT was calculated by plotting the residual of the individual relationship between energy expenditure and physical activity in time, as measured over 30-min intervals from a 24-h observation in a respiration chamber [ 10 ]. Subjects were 17 females and 20 males. The level of resting metabolic rate after waking up in the morning, and directly before the first meal, was defined as basal metabolic rate. Resting metabolic rate did not return to basal metabolic rate before lunch at 4 h after breakfast, or before dinner at 5 h after lunch. Overnight, basal metabolic rate was reached at 8 h after dinner consumption. Figure 1 The mean pattern of diet induced thermogenesis throughout the day, calculated by plotting the residual of the individual relationship between energy expenditure and physical activity in time, as measured over 30-min intervals from a 24-h observation in a respiration chamber. Subjects were 17 females and 20 males [10]: -----, level of basal metabolic rate; arrows, meal times. Fifteen studies on DIT with information on energy intake, on diet composition and on the postprandial measurement period were selected from literature (Table 1 ). Five studies compared DIT, as measured with the same protocol in the same subjects, for two or more diets with a different composition. For alcohol, there was a tendency for an increased DIT, from 7.2 to 8.6 % of the energy content of the meal, when 22% of the energy content of a meal was exchanged for an alcoholic aperitif [ 11 ]. In a second study, with a similar energy exchange with alcohol, there was a significant increase in DIT, from 7.1 to 9.0 % of the energy content of the meal [ 12 ]. For protein, there was a tendency for and increased DIT, from 7.1 to 8.3% when 20 en% of the meal was exchanged with protein [ 12 ]. In a second study, with a similar energy exchange with protein, there was a significant increase in DIT, from 10.5 to 14.6 % of the energy content of the meal [ 6 ]. For carbohydrate and fat, one study showed no effect [ 12 ], one study showed an increase after the exchange of 65 en% fat for carbohydrate [ 13 ], and one study showed the opposite, a decrease after the exchange of 28 en% fat for carbohydrate [ 14 ]. Table 1 Diet composition, expressed as the energy contribution of carbohydrate, protein, fat and alcohol (en% C/P/F/A) of food intake, and diet-induced thermogenesis (DIT), measured as the increase in energy expenditure above basal fasting level over the presented time interval and expressed as a percentage of the energy content of the food ingested (% intake). Reference subjects (n) diet (en% C/P/F/A) intake (MJ) time (h) DIT (% intake) [3] 10 57/10/33/0 1.9 4 7.1 [11] 12 45/10/45/0 2.5 4 7.2 12 35/8/35/22 2.5 4 8.6 [24] 6 0/0/0/100 0.9 4 17.1 [5] 471 50/20/30/0 9.4 24 18 [25] 6 73/11/16/0 2.8 4 4.2 [26] 6 65/10/25/0 2.5 5 6.5 [13] 18 80/18/2/0 2.2 4 4.0 a 18 15/18/67/0 2.2 4 5.0 b [27] 12 45/15/40/0 3.1 5.5 8.3 [6] 8 30/10/60/0 8.9 24 10.5 a 8 60/30/10/0 8.9 24 14.6 b [28] 12 45/15/40/0 3.6 5.5 7.1 [29] 24 62/27/11/0 3.8 5 8.1 [14] 12 40/12/48/0 2.5 5 4.3 a 12 68/12/20/0 2.5 5 6.5 b [30] 14 42/15/43/0 2.5 5 5.8 [31] 13 80/17/3/0 3.2 4 5.2 [12] 19 37/32/31/0 2.8 5 8.3 19 65/12/24/0 2.8 5 7.1 a 19 24/12/65/0 2.8 5 7.1 a 19 43/12/24/23 2.8 5 9.0 b For a comparison of DIT between studies as a function of the nutrient composition of the test food consumed, the energy content of the test food was divided by the length of the measurement interval after food consumption and expressed in MJ/h. Only three of the 22 studies presented in table 1 included alcohol as a nutrient and were excluded. In a regression analysis of the remaining 19 studies, the protein fraction of the food came out as significant determinant of DIT. An increase in the protein fraction of one % resulted in an increase of DIT with 0.22 ± 0.42 % (p < 0.05). Discussion The main determinant of DIT is the energy content of the food, followed by the protein fraction of the food. The thermic effect of alcohol is similar to the thermic effect of protein. Diet induced thermogenesis is related to the stimulation of energy-requiring processes during the post-prandial period. The intestinal absorption of nutrients, the initial steps of their metabolism and the storage of the absorbed but not immediately oxidized nutrients [ 15 ]. As such, the amount of food ingested quantified as the energy content of the food is a determinant of DIT. The most common way to express DIT is derived from this phenomenon, the difference between energy expenditure after food consumption and basal energy expenditure, divided by the rate of nutrient energy administration [ 16 ]. Theoretically, based on the amount of ATP required for the initial steps of metabolism and storage, the DIT is different for each nutrient. Reported DIT values for separate nutrients are 0 to 3% for fat, 5 to 10% for carbohydrate, 20 to 30% for protein [ 16 ], and 10 to 30% for alcohol [ 6 ]. In healthy subjects with a mixed diet, DIT represents about 10% of the total amount of energy ingested over 24 h. When a subject is in energy balance, where intake equals expenditure, DIT is 10% of daily energy expenditure. Of the studies presented in table 1 , most reported a DIT value below 10% of the energy content of the food ingested. The studies reporting a DIT value below 10% measured DIT as the increase in energy expenditure above basal fasting level over an interval of 4 to 5.5 h after the meal. The studies with a higher value included a study with pure alcohol consumption and the studies where DIT was measured over 24 h in a respiration chamber. In the respiration chamber studies, DIT values were calculated as the increase in energy expenditure above sleeping metabolic rate while the other studies reported DIT as the increase in energy expenditure above basal metabolic rate. Basal metabolic rate is about 5% higher than sleeping metabolic rate [ 17 ]. After correction of the DIT values based on sleeping metabolic rate to the increase in energy expenditure above basal metabolic rate, the chamber values are close to the values of 10% of daily energy intake. The higher DIT value of alcohol and protein compared with carbohydrate and fat has implications for the effect of these nutrients on energy balance. However, the main effect on energy balance does not seem to be primarily linked to the lower bioavailability of alcohol-and protein energy than that of fat and carbohydrate. Alcohol energy is largely additive to the normal diet but does not seem to affect energy balance positively [ 18 ]. Protein plays a key role in food intake regulation through satiety related to DIT [ 19 ]. Alcohol forms a significant component of many diets and it supplements rather than displaces daily energy intake. Alcohol consumption as an aperitif has even been shown to result in a higher subsequent intake with no intake compensation afterwards [ 20 ]. Yet, alcohol intake does not systematically increase body weight. In a recent study, it was shown that subjects with higher alcohol consumption are habitually more active [ 21 ]. This may be one explanation for the lack of increasing body weight through additional energy intake from alcohol. The main effect of protein on energy balance is thought to be DIT related satiety. Satiety scores were higher during meals with a high-protein/high-carbohydrate diet, as well as over 24 h, than with a high-fat diet [ 22 ]. The observed DIT related satiety might be ascribed to the high protein rather than the high carbohydrate content of the diet. Postprandial thermogenesis was increased 100% on a high-protein/low-fat diet versus a high-carbohydrate/low-fat diet in healthy subjects [ 23 ]. The DIT increases body temperature, which may be translated into satiety feelings. High-protein diets are favored for weight maintenance, also after weight loss, by favoring maintenance or regain of fat-free mass, by reducing the energy efficiency through a higher thermogenesis, and by reducing intake through an increased satiety [ 19 ]. In conclusion, the main determinants of diet-induced thermogenesis are the energy content and the protein-and alcohol fraction of the diet. Protein plays a key role in body weight regulation through satiety related to diet-induced thermogenesis.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524030.xml
212691	The Roles of APC and Axin Derived from Experimental and Theoretical Analysis of the Wnt Pathway	Wnt signaling plays an important role in both oncogenesis and development. Activation of the Wnt pathway results in stabilization of the transcriptional coactivator β-catenin. Recent studies have demonstrated that axin, which coordinates β-catenin degradation, is itself degraded. Although the key molecules required for transducing a Wnt signal have been identified, a quantitative understanding of this pathway has been lacking. We have developed a mathematical model for the canonical Wnt pathway that describes the interactions among the core components: Wnt, Frizzled, Dishevelled, GSK3β, APC, axin, β-catenin, and TCF. Using a system of differential equations, the model incorporates the kinetics of protein–protein interactions, protein synthesis/degradation, and phosphorylation/dephosphorylation. We initially defined a reference state of kinetic, thermodynamic, and flux data from experiments using Xenopus extracts. Predictions based on the analysis of the reference state were used iteratively to develop a more refined model from which we analyzed the effects of prolonged and transient Wnt stimulation on β-catenin and axin turnover. We predict several unusual features of the Wnt pathway, some of which we tested experimentally. An insight from our model, which we confirmed experimentally, is that the two scaffold proteins axin and APC promote the formation of degradation complexes in very different ways. We can also explain the importance of axin degradation in amplifying and sharpening the Wnt signal, and we show that the dependence of axin degradation on APC is an essential part of an unappreciated regulatory loop that prevents the accumulation of β-catenin at decreased APC concentrations. By applying control analysis to our mathematical model, we demonstrate the modular design, sensitivity, and robustness of the Wnt pathway and derive an explicit expression for tumor suppression and oncogenicity.	Introduction Considerable effort employing biochemistry, genetics, and pharmacology has been invested in identifying the web of interactions that characterize signal transduction pathways in metazoan organisms. Several conclusions can be drawn from these efforts. Despite the large number of receptors, ligands, and downstream targets, the number of signal transduction pathways in metazoans is relatively small, arguably less than 20 ( Gerhart 1999 ). This limited diversity occurs despite large numbers of different organisms, cell types, states of growth, and differentiation, as well as sexual dimorphism in biology. Remarkably, these pathways are highly conserved, some among all eukaryotes, most among all metazoans. Whereas signaling pathways differ in detail, it is not clear whether these differences are functionally significant. Conservation in the face of diversity of function raises the question of whether the behaviors of the pathway are in reality as similar as they seem when one compares more quantitative aspects of the signals and responses, such as amplitude, duration, and flux ( Heinrich et al. 2002 ). Finally, the structure and design of the pathways are themselves a mystery. Is the structure of these conserved pathways so deeply embedded in other conserved process that it is difficult to change any interaction, or does conservation imply continuous selection for function ( Gerhart and Kirschner 1997 )? Many of these questions require a more quantitative understanding of the behavior of signaling pathways. Such information is rarely available. Most mathematical models have to be satisfied with a general conceptual understanding and are seldom testable, as most parameters must be assumed or inferred. It is partly for this reason that such theoretical efforts up to now have had limited impact on experimentalists, who prefer powerful qualitative tools to construct logical and formal models of pathway structures. Mathematical modeling is more advanced for metabolic networks, where the pathways have been known for more than a half-century and where more kinetic data have been available, including more recent data on in vivo dynamics ( Heinrich and Schuster 1996 ). To develop a better quantitative understanding of a signal transduction pathway, we have recreated a more accessible system for biochemical study. The Wnt signaling pathway downstream of its immediate cytoplasmic mediator, Dishevelled (Dsh), can be reconstituted in frog egg extracts. The readout of the pathway is the rate of degradation of the transcriptional coactivator, β-catenin. We chose the Wnt pathway because it is active in the early Xenopus embryo, it is widely used in many different contexts in development, and it is also very important in human cancer. Although the design features of the Wnt pathway are highly conserved in evolution, it is not clear what purposes those features serve. This paper is in part an answer to that question. The pivotal player in Wnt signaling is the scaffold protein axin, which is required for the constitutive degradation of β-catenin. Axin coordinates the assembly of a large complex that includes the glycogen synthase kinase 3β (GSK3β); another scaffold protein, adenomatous polyposis coli (APC); and the negative regulators Dsh and GSK3β-binding protein (glycogen synthase kinase-binding protein [GBP]/Frat). Binding of Wnt to its receptor, Frizzled, activates Dsh through an as-yet-unknown process. In the absence of Wnt, GSK3β bound to axin phosphorylates β-catenin bound to both axin and APC. Phosphorylated β-catenin is a substrate for ubiquitination and subsequent degradation through the F-box protein β-TRCP, which is part of an SCF ubiquitin ligase complex. In the presence of the Wnt signal, the activated Dsh protein binds to axin and, through bound GBP, inhibits β-catenin phosphorylation; this inhibits its ubiquitination and consequent degradation. The buildup in β-catenin in the presence of a Wnt signal leads to transcription of specific genes. Numerous questions arise from this general model. Why are two scaffold proteins, APC and axin, both necessary? Do their roles differ? Recently it has been discovered that axin, like β-catenin, is an unstable protein ( Yamamoto et al. 1999 ; Tolwinski et al. 2003 ). In recent work (unpublished data), we have further described the conditions under which axin is unstable. We ask here what role axin instability plays in the behavior of the Wnt pathway and in the responsiveness of the pathway to the Wnt signal? Beyond these mechanistic questions are important biological ones that lie beyond the scope of this work but that may be raised by some of the findings here. For example, mutations in APC seem to play a particularly important role in colorectal cancer; is the peculiar sensitivity to APC mutations in the colonic epithelium understandable in terms of how the pathway performs in that tissue? Similarly, GSK3β is also essential but not commonly mutated in colorectal cancer; why is that the case? No one has purified a discrete complex containing all the major players in the Wnt pathway arrayed on the axin–APC scaffolds, suggesting that the pathway might be affected by nonproductive titration of components by subcomplexes. If this is a problem, how is it avoided? The Wnt pathway is likely present in all cell types, and yet several of its constituents are used in other pathways; how is crosstalk or interference in other pathways avoided? As we set out to produce a realistic kinetic model of the Wnt pathway, we encountered other questions. For example, quantitative measurements led to the unusual finding that axin is present at very low concentrations. Is there a satisfactory explanation of this fact and of other, previously unexplained, features of the pathway? We are aware that some of the answers to specific questions could lie in unknown components or unknown interactions among known components. We were under no illusions that we could accommodate all known interactions in a model at this time or that we already knew all we need to know to construct such a model. For this reason we have asked a more modest question—whether the properties of the core pathway, as presently understood, can provide insight into important questions not yet answered or not yet clearly raised. To answer such questions, we collected what we initially thought were sufficient quantitative data on rates, affinities, and fluxes to derive a reference state model of the Wnt pathway in this system. The provisional reference state model reflected most of the known properties of the system, but from this model we identified several rates and affinity constants whose values were critical to the behavior of the model. We then went back and measured these. Thus this paper contains a reference model, a large number of experimental measurements needed to define this model, theoretical predictions, and experimental tests of those predictions, where possible. A general test of the validity of the model is its predictive ability under a wide range of conditions. From this analysis, several unexpected properties emerged with significance for understanding the biological function of the Wnt pathway. Results A Proposed Kinetic Pathway The model was based on the reaction scheme shown in Figure 1 . A few steps are labeled, such as the synthesis of axin and β-catenin, the degradation of axin, the axin-independent (basal) and axin-dependent degradation of β-catenin, as well as the critical cycle involved in the phosphorylation of β-catenin for degradation (Destruction Core Cycle). The output is the formation of the β-catenin/T-cell factor (TCF) complex and the input is the Wnt signal. Although many proteins interact with the Wnt pathway, we have focused only on core components known to be necessary for mediating a Wnt signal in most contexts. These core proteins include GSK3β, protein phosphatase 2A (PP2A), β-catenin, APC, axin, Dsh, TCF, and Wnt. The reactions incorporated into our model include protein synthesis/degradation, protein phosphorylation/dephosphorylation, and the assembly/disassembly of protein complexes ( Figure 1 , solid arrows). Reactions mediated by proteins that activate a process are represented with broken arrows: (1) activation of Dsh by Wnt (step 1), (2) activation of the release of GSK3β from APC/axin/GSK3β by Dsh (step 3), and (3) activation of APC-dependent axin degradation (step 15). The reactions and components in blue are concerned with additional features of the pathway, as discussed below. Figure 1 Reaction Scheme for Wnt Signaling The reaction steps of the Wnt pathway are numbered 1 to 19. Protein complexes are denoted by the names of their components, separated by a slash and enclosed in brackets. Phosphorylated components are marked by an asterisk. Single-headed solid arrows characterize reactions taking place only in the indicated direction. Double-headed arrows denote binding equilibria. Blue arrows mark reactions that have only been taken into account when studying the effect of high axin concentrations. Broken arrows represent activation of Dsh by the Wnt ligand (step 1), Dsh-mediated initiation of the release of GSK3β from the destruction complex (step 3), and APC-mediated degradation of axin (step 15). The broken arrows indicate that the components mediate but do not participate stoichiometrically in the reaction scheme. The irreversible reactions 2, 4, 5, 9–11, and 13 are unimolecular, and reactions 6, 7, 8, 16, and 17 are reversible binding steps. The individual reactions and their role in the Wnt pathway are explained in the text. The centerpiece of the model is the formation of the unstable core complexes involved in β-catenin phosphorylation and subsequent destruction. In addition to β-catenin, this set of complexes contains GSK3β and the scaffold proteins APC and axin. The complexes assemble in several steps: (1) binding of axin to APC (forward reaction of step 7); (2) binding of GSK3β (forward reaction of step 6); (3) phosphorylation of axin and APC by GSK3β (step 4). Dephosphorylation of the core complex (step 5) is mediated by PP2A. The first step in β-catenin degradation is its binding to APC/axin/GSK3β (step 8), after which it is phosphorylated by GSK3β (step 9) and released from the complex (step 10). Our model assumes that the phosphorylation of β-catenin by GSK3β is negligible in the absence of axin. Indeed, recent work indicates that axin stimulates the phosphorylation of β-catenin by GSK3β at least 24,000-fold ( Dajani et al. 2003 ). Free, phosphorylated β-catenin is rapidly polyubiquitinated and degraded by the SCF complex and the proteasome, respectively (step 11). The dynamic properties of the model, such as the flux through the pathway, are also affected by binding of β-catenin to other partners, such as TCF (step 16) and free APC (step 17). In special cases (high axin concentrations), the flux through the system is affected by the binding of axin to GSK3β (step 19) as well as β-catenin (step 18). We have previously shown experimentally that TCF reduces the rate of β-catenin degradation ( Lee et al. 2001 ). Turnover of β-catenin (steps 11, 12, and 13) and axin (steps 14 and 15) are included in our model, but since biochemical experiments in Xenopus egg extracts indicate that the turnover of GSK3β, Dsh, and TCF is relatively slow (no detectable degradation after 3 h at room temperature; unpublished data), the synthesis and degradation of these proteins are not explicitly modeled. The activation of the pathway in vivo, which results in the stabilization of β-catenin, is initiated by binding of Wnt ligands to Frizzled receptors and the subsequent transition of Dsh from its inactive form (Dsh i ) to its active form (Dsh a ). Since these events are still poorly defined, both processes have been combined in step 1. Interaction of Dsh a with the nonphosphorylated complex APC/axin/GSK3β (step 3) activates the release of GSK3β . This latter process requires the activity of the GBP/Frat (not shown on our diagram). Deactivation of Dsh a occurs through an as-yet-unidentified mechanism (step 2). Analytical Description The mathematical analysis is based on a series of balance equations that describe the concentrations and complexes of proteins in the Wnt pathway, as depicted in Figure 1 . The set of variables and the set of 15 differential equations we obtained are given in Table S1 , and Dataset S1 , respectively (Equations [A-1]–[A-15]). Stimulation of the pathway by Wnt is described by a time-dependent function, Wnt(t) . Since Dsh, TCF, and GSK3β are degraded very slowly, we assume that their concentrations remain constant throughout the timecourse of a Wnt signaling event. The conservation equations for Dsh, TCF, and GSK3β are as follows: Symbols with the superscript "0" denote total concentrations. Since the concentration of axin is very low (see below) compared to the concentration of GSK3β, we replaced Equation (3) with the simple relationship GSK3β 0 = GSK3β . Similarly, we omitted the concentration of complexes containing axin in the conservation relationship for APC, which leads to the following equation: We will, however, take into account the contribution of axin-containing complexes for GSK3β and APC conservation equations when we later consider the effect of large increases in axin concentration. The simplest possible equation was chosen to describe the kinetics of each individual reaction. Synthesis of β-catenin and axin are described by constant rates ν i . Unimolecular reactions are assumed to be irreversible and are described by linear rate equations ν i = k i · X j , where k i denotes the first-order rate constant and X j denotes the concentration of the reactants. Reversible binding steps (double-headed arrows in Figure 1 ) are described by the equation ν i = k +i X j Y l − k −i ( X j . Y l ), where X j and Y l denote the concentrations of the binding partners and ( X j . Y l ) the concentration of their complex. The Dsh-mediated release of GSK3β from the destruction complex is described by an irreversible reaction that is bimolecular in the concentrations of Dsh and the degradation complex. The model is simplified by assuming that the reversible binding steps between axin, β-catenin, APC, and TCF are very fast, such that the corresponding protein complexes are in rapid equilibrium, so that only the dissociation constants K i = k − i / k + i are considered in the kinetic description of these steps. The conservation equations and the binding equilibria reduce the number of independent dynamic variables. Accordingly, the original set of 15 differential equations is transformed into a set of only seven ordinary differential equations coupled to four conservation equations and four relationships for binding equilibria. For a detailed mathematical description of the model and the procedure for reducing the number of systems variables, see Dataset S1 . Experimental Evaluation of the Reference and Stimulated States We define the reference state as the absence of Wnt signaling ( Wnt = 0). In this unstimulated stationary state, Dsh is inactive and does not affect the degradation complex. β-Catenin concentration is kept low by continuous phosphorylation and degradation. The reference state can be characterized by the special values for its rate constants, its equilibrium constants, and its conservation quantities. If one can obtain values for all of these system parameters, the model equations should allow for a straightforward calculation of the variables in the reference state. Currently, we have experimental data for many of these parameters (see below). For the remaining system parameters that were not directly measured, we were able to derive numbers based on experimental data of steady-state concentrations and fluxes. A number of parameters were set such that the results of the model were in agreement with previous experimental data, specifically with the experimentally determined rate of β-catenin degradation ( Salic et al. 2000 ; Lee et al. 2001 ). Finally, a few parameters had to be estimated; the constraint used was that the resulting model should be compatible with the steady-state and flux values. Table 1 lists the numeric values of all of the input quantities of the model. These quantities are either specific parameters, such as dissociation constants, or systemic properties, such as steady-state concentrations or fluxes, from which the other parameters have been derived. Both types of input quantities include experimental data as well as estimated values. The specific numerical values affect the model to differing degrees. In a later section, we analyze the effects of changing the values of the parameters around their reference numbers. The types of input data used for our modeling can be divided into five groups. Table 1 Numeric Values of Input Quantities of the Model for the Reference State The data are grouped into concentrations of pathway components, dissociation constants of protein complexes, concentration ratios, fluxes and flux ratios, and characteristic times of selected processes. Experimental evidence for these data is discussed in the text. From these data, the following rates and rate constants are calculated: ν 12 = 0.42 nM · min −1 (rate of β-catenin synthesis), ν 14 = 8.2 · 10 −5 · nM min −1 (rate of axin synthesis), k 4 = 0.27 min −1 , k 5 = 0.13 min −1 , k 6 = 9.1 · 10 −2 nM −1 · min −1 , k − 6 = 0.91 · nM −1 · min −1 , k 9 = 210 min −1 , k 10 = 210 min −1 , k 11 = 0.42 min −1 , k 13 = 2.6 · 10 −4 min −1 , k 15 = 0.17 · min −1 . See Table S2, found at http://dx.doi.org/10.1371/journal.pbio.0000010.st002 , for more precise numbers used in the calculations Bold: Measured values, Italics : Estimated values The first group of input data contains both total concentrations ( Dsh 0 , APC 0 , TCF 0 , and GSK3β 0 ) and steady-state concentrations ( Axin 0 , β-catenin 0 , β-catenin ). The total concentrations of Dsh, TCF, GSK3β, axin, β-catenin, and APC in Xenopus egg extract were determined experimentally using Western blot analysis by comparing the intensity of the signal to that of known amounts of recombinant protein. The concentration of phosphorylated β-catenin w as estimated because we have not been able to directly determine its level in extracts. However, we estimate that this value is small compared to that of nonphosphorylated β-catenin for the following reasons: (1) Addition of axin to Xenopus extracts dramatically increases the rate of β-catenin degradation. Since the role of axin is to promote phosphorylation of β-catenin, which is subsequently degraded, this would suggest that normally a significant pool of β-catenin exists in the nonphosphorylated form. (2) Western blot analysis of Xenopus extracts demonstrates that only a small percentage (<10%) of total β-catenin can be detected as migrating with a slower mobility, which likely represents the phosphorylated form of β-catenin. The second group of input data was experimentally obtained from measurements of rates of dissociation of protein complexes. Binding constants were calculated based on the assumption that association rates approached that of the diffusion limits for a typical protein in an aqueous solution. The ratio K 17 / K 8 = 10 of the dissociation constants characterizing the binding of β-catenin to APC and APC/axin/GSK3β, respectively, is based on previous experimental results demonstrating that β-catenin has a 10-fold lower affinity for nonphosphorylated compared to phosphorylated APC ( Salic et al. 2000 ). In addition, we have shown experimentally (unpublished data; see Materials and Methods) that phosphorylated β-catenin dissociates from axin more rapidly (reaction 10) than nonphosphorylated β-catenin. Once phosphorylated, β-catenin will thus dissociate from the axin complex and undergo polyubiquitination and proteolysis. The third group of input data consists of the two concentration ratios in the Destruction Core Cycle for complexes that either contain or lack β-catenin. The concentration ratio for the complexes that lack β-catenin is represented by the ratio of its phosphorylated versus nonphosphorylated forms and reflects the relative activities of its kinase(s) and phosphatase(s), respectively. By contrast, the concentration ratio of the two β-catenin-containing degradation complexes represents the relative activities of β-catenin phosphorylation and the rate of release of phosphorylated β-catenin from the complex. These parameters were chosen rather arbitrarily to indicate roughly equal kinase and phosphatase activities and yielded realistic values for the overall fluxes, given the known concentrations and kinetic rate constants. The fourth group of data includes the steady state flux ν 11 for the degradation of β-catenin via the Wnt pathway and the flux ratio ν 13 / ν 11 describing the extent to which β-catenin is degraded via non-Wnt mechanisms (e.g., via Siah-1 and presenilin [ Liu et al. 2001 ; Matsuzawa and Reed 2001 ; Kang et al. 2002 ]). We have now measured this Wnt pathway–independent degradation in Xenopus extracts (see Materials and Methods; value shown in Table 1 ). The final group of input data consists of the characteristic time constant (τ) of selected processes. This is the time it takes for the concentration to drop to 1/e of its initial value. These characteristic times include τ K . P = 1/( k 4 + k 5 ) for the kinase/phosphatase cycle that mediates phosphorylation/dephosphorylation of both APC and axin in the degradation complex (steps 4 and 5), τ GSK3β . ass = 1 / ( k 6 GSK3β + k −6 ) for the binding equilibrium of GSK3β with the APC/axin complex (step 6), and τ ax . deg for axin degradation (step 15). Values for the rate of axin degradation were determined directly from experiments performed in Xenopus egg extracts (unpublished data). Experiments to determine the rate of APC and axin dephosphorylation (τ K . P ≈ 2.5 min) were performed using in vitro 32 P-labeled recombinant APC and axin. Radiolabeled proteins were added to Xenopus egg extracts, and the loss of radioactivity over time was assessed by SDS-PAGE and autoradiography ( Salic et al. 2000 ). The legend to Table 1 contains the values of rate constants calculated from the input quantities using the described system of equations. The values of all variables in the reference state are listed in the first column of Table 2 . These values represent the steady state solutions of system equations using the data in Table 1 as input quantities with the value of Wnt set at Wnt = 0. Table 2 Steady-State Concentrations of Pathway Compounds in the Reference State and in the Standard Stimulated State Calculation of the concentrations is performed by the systems equations (see Dataset S1) under steady-state conditions. For the reference state ( W = 0), the corresponding parameters given in Table 1 are used. Values marked by “#” represent steady-state concentrations, which also appear in Table 1 . The additional input quantities required for the calculation of the standard stimulated state ( W = 1) are taken as follows: Dsh a / Dsh i = k 1 / k 2 = 10, ν −6 / ν 3 = k −6 / ( k 3 Dsh a ) = 0.2, and τ Dsh . act = 1 / ( k 1 + k 2 ) = 5 min. This yields the following values for rate constants: k 1 = 0.18 min −1 , k 2 = 1.8 · 10 −2 min −1 , k 3 = 5.0 · 10 −2 nM −1 · s −1 Using the reference state as a starting point, we consider other stationary states that are attained when the pathway is permanently stimulated. To describe the strength of Wnt stimulation, we introduce a dimensionless quantity W = Wnt/Wnt 0 that represents the ratio of the Wnt concentration with respect to its concentration Wnt 0 in a “standard” stimulated (signaling) state. W = 0 and W = 1 characterize the reference state and a standard stimulated state, respectively, with the hyperstimulated state defined as W > 1. In order to calculate concentrations in the standard stimulated state, additional input quantities are required. These include the ratio of the active and inactive forms of Dsh ( Dsh a /Dsh i ), the relation between non-Dsh-mediated and Dsh-mediated release of GSK3β from the destruction complex (the flux ratio ν − 6 / ν 3 ), and the characteristic time for the Dsh activation/inactivation cycle (τ Dsh . act ). These values are not at present measurable. The values for these input quantities are listed in the legend of Table 2 . In a later section, we analyze the effects of changes in these additional input quantities. By setting W = 1 and fixing all other parameters, we arrive at steady-state solutions of the systems equations (see Dataset S1 , Equations. [A-1]–[A-15]), which yield the numerical variables for the standard stimulated state (listed in the second column of Table 2 ). A comparison of this state with the reference state shows that the concentration of free nonphosphorylated β-catenin increases by a factor of approximately 6, from 25 to 153 nM. Upon Wnt stimulation, the free phosphorylated β-catenin concentration decreases by 8%, from 1 nM to 0.92 nM. The increase in β-catenin levels reflects the decrease in its degradation caused by the reduction in the ability of GSK3β to phosphorylate it. The concentration of the β-catenin/TCF complex increases by a factor of 1.8. The large increase in β-catenin concentration shifts the binding equilibrium between APC and β-catenin and the concentration of free APC falls slightly. Total axin concentration decreased by a factor of 2.7 upon Wnt stimulation since addition of Dsh decreases the concentration of the various axin containing complexes. Remarkably, the steady-state concentration of free axin is constant during the transition from W = 0 to W = 1. This is due to the fact that under steady-state conditions, the rate of axin synthesis equals its degradation; the rate of synthesis ( ν 14 ) is a fixed value and the rate of degradation depends solely on the concentration of free axin (and independent of other parameters such as binding constants and strength of the Wnt signal). As expected, simulations of increasing Wnt activation (0 ≤ W ≤ 1.4) on the steady-state concentrations of β-catenin and axin reveal a nearly hyperbolic saturation of increasing concentrations of nonphosphorylated and total β-catenin with increasing strength of Wnt stimulation. Furthermore, Wnt stimulation affects the steady-state concentrations of axin and β-catenin in an opposite direction (see Figure S1 ). β-Catenin Degradation: Comparison of Theory and Experiment To test whether the mathematical model represented the Wnt pathway under a variety of conditions, we ran through a series of simulations, all of which used the same set of parameters. From these we calculated simulated timecourses for β-catenin degradation under a range of different conditions (increased axin concentration, increased Dsh a concentration, inhibition of GSK3β, increased TCF concentration) ( Figure 2 A). We then tested the results using the previously described biochemical system ( Salic et al. 2000 ; Lee et al. 2001 ), adding purified proteins or compounds at t = 0 ( Figure 2 B). Simulations and experimental results are each shown as plots of total β-catenin concentration versus time. The agreement between theory and experiment is excellent. Figure 2 Kinetics of β-Catenin Degradation: Simulation and Experimental Results (A) Simulated timecourses of β-catenin degradation. The straight line for t < 0 corresponds to the reference state of β-catenin using the parameters given in the legends of Table 1 and 2. In vitro conditions are simulated by switching off synthesis of β-catenin and axin ( ν 12 = 0, ν 14 = 0 for t ≥ 0). Curve a: reference case (no addition of further compounds); curve b: addition of 0.2 nM axin; curve c: addition of 1 μM activated Dsh (deactivation of Dsh was neglected, k 2 = 0); curve d: inhibition of GSK3β (simulated by setting k 4 = 0, k 9 = 0); curve e: addition of 1μM TCF. Addition of compounds (axin, Dsh, TCF) and inhibition of GSK3β was performed at t = 0. (B) Experimental timecourse of β-catenin degradation in Xenopus egg extracts in the presence of buffer (curve a′), axin (curve b′: 10 nM), Dsh (curve c′: 1 μM), Li + (curve d′: 25 mM), or Tcf3 (curve e′: 1 μM). The straight line for t < 0 represents the reference state. The simulated reference state curve ( Figure 2 A, curve a) for β-catenin degradation is calculated for t > 0, at which there is an absence of protein synthesis for axin ( ν 14 = 0) and β-catenin ( ν 12 = 0). This reference curve is in close agreement with our experimental data ( Figure 2 B, curve a′) with identical half-lives for β-catenin degradation (theoretical value of t ½ = 60.2 min versus experimental value of t ½ = 60 min). We examined a new state, where we have increased the amount of endogenous axin (0.02 nM) by 0.2 nM. As shown in Figure 2 A, curve b, the additional axin markedly accelerated β-catenin degradation ( t ½ = 11.8 min) in agreement with the experimentally obtained values ( Figure 2 B, curve b′; t ½ = 12 min). Theoretically, the effect of axin on β-catenin degradation is primarily due to the large concentration difference between the two scaffold proteins, APC and axin. Owing to the high concentration of APC, an increase in axin concentration results in a sharp increase in the concentration of the APC/axin complex, thereby accelerating β-catenin binding to the destruction complex. Curve d in Figure 2 shows the effect of inhibiting GSK3β on β-catenin degradation. This effect is produced in the simulation by inhibiting GSK3β activity (steps 4 and 9). Only a small fraction of β-catenin (phosphorylated β-catenin) is available for degradation after complete inhibition of β-catenin phosphorylation (step 9), so inhibition is rapid. This is in complete agreement with our experimental data in which degradation is essentially blocked after inhibiting GSK3β activity by lithium ( Figure 2 B, curve d′). Curve e in Figure 2 A predicts that β-catenin degradation is strongly inhibited after the addition of 1 μM TCF. Previously we have shown that β-catenin is sequestered by TCF, thereby resulting in a significant decrease in free β-catenin ( Lee et al. 2001 ). The addition of TCF would be expected to decrease the rate of β-catenin phosphorylation (step 9) and subsequently β-catenin degradation. This is also seen experimentally ( Figure 2 B, curve e′). The immediate inhibition by LiCl is in contrast with the action of Dsh that inhibits only after a significant delay. We were intrigued by the theoretical biphasic degradation curves of β-catenin in the presence of Dsh a , as well as the experimental support for it ( Figure 2 A and 2B, curves c and c′). In both cases, there is an initial rapid decrease in β-catenin in the first 30 min to 1 h, followed by a much slower decrease. Such a feature should allow us to distinguish mechanistic details of complex formation. Experimentally, the biphasic nature of Dsh activity is not due to a delay in Dsh activation upon its addition to the Xenopus extracts since we see the same effect with Dsh protein that has been “activated” with extracts prior to its use in our degradation assay. As shown in Table 1 , the characteristic time τ K . P of phosphorylation and dephosphorylation of APC and axin in the destruction complex is relatively slow (2.5 min), and it therefore takes 5 min for 75% of the complex to be dephosphorylated. If Dsh a acted only on the dephosphorylated complex (through step 3) to remove GSK3β and thus block phosphorylation of the complex, then we would predict the biphasic kinetics shown in Figure 2 A, curve c. These data suggest that Dsh inhibits the phosphorylation of the scaffold complex by GSK3β, but does not inhibit the phosphorylation of β-catenin. When Dsh binds, the complex can go around many times binding and phosphorylating β-catenin before it dissociates and is inhibited by Dsh. One hour after the addition of Dsh, β-catenin degradation is significantly inhibited due to the removal of a significant pool of GSK3β from the degradation complex over time (through the action of Dsh). As a result, the scaffold protein axin is dephosphorylated by the phosphatase (step 5) that remains bound to the degradation complex. Dephosphorylated axin is rapidly ubiquitinated and degraded when the β-catenin degradation normally stops. The small decrease in β-catenin levels in Figure 2 , curve c, after a 1 h incubation with Dsh, is due to degradation of β-catenin via nonWnt pathway mechanisms (see Table 1 ) that we have incorporated into our model. To test this prediction beyond consistency with experimental data, we performed an experiment in which Dsh was either preincubated with extract before or added at the same time as radiolabeled β-catenin ( Figure 3 ). If β-catenin and Dsh are added at the same time, there is an initial rapid loss of β-catenin ( Figure 3 , curve b) followed by pronounced inhibition of degradation after 1 h. This initial rapid loss is consistent with Dsh acting on a subpopulation of degradation complexes (presumably the unphosphorylated forms). Strikingly, preincubation with Dsh prior to the addition of radiolabeled β-catenin ( Figure 3 , curve a) results in immediate action of Dsh. We interpret this result to simply reflect the fact that over time in the preincubated extract Dsh can remove GSK3β from the degradation complexes, thereby enhancing the activity of the phosphatase and, as a result, promoting the degradation of axin and inhibition of β-catenin degradation. The small decrease in β-catenin levels at t > 2 h in both curves a and b again suggests the existence of a slow degradation process mediated by non-Wnt pathway mechanisms. Figure 3 Preincubation of Dsh in Xenopus Egg Extracts Abolishes the Lag in Dsh Activity Labeled β-catenin was incubated in Xenopus extracts on ice 30 min prior to (B) or 30 min after (A) the extract had been preincubated with 1 μM Dsh. No degradation of the labeled β-catenin was detected while the reactions were on ice. The reactions were started by shifting to 20°C. Clues to Axin Activity from Its Very Low Cellular Concentration In establishing quantities for our model in Table 1 , we found that the axin concentration (20 pM) is much lower than the concentration of the other major components (β-catenin, 35 nM; APC, 100 nM; Dsh, 100 nM; and GSK3β, 50 nM). This unusual finding suggests that the function of the Wnt signaling system may actually depend on a low axin concentration. Our theoretical predictions for the effects of axin, GSK3β, and Dsh on the half-lives of β-catenin are shown in Figure 4 A and 4B, respectively. At zero concentration of Dsh, doubling the concentration of axin (from the reference state, indicated as 0, to a state where the concentration has been increased by 0.02 nM) causes a 50% drop in the half-life of β-catenin. By contrast, a doubling of the GSK3β concentration only decreases the half-life of β-catenin by 10%. The small effect of GSK3β is predicted to be due to the fact that only a limited amount of axin can be recruited to the degradation complex through binding to additional GSK3β. On the other hand, increased axin concentrations are immediately translated into an increased concentration of the destruction complex, because the concentrations of APC and GSK3β are high. Changing the concentration of either GSK3β or of axin should also change the amount of Dsh a required to inhibit β-catenin degradation, but the pathway is much more sensitive to axin concentration than it is to GSK3β concentration. In the presence of high concentrations of axin, the effect of Dsh a should be blocked; high concentrations of axin will lead to high concentrations of the phosphorylated destruction complex no matter what level of Dsh a activity is present. High levels of the destruction complex will require even higher levels of Dsh to overcome the inhibition. The interaction between Dsh a and GSK3β is similar in principle: Dsh-mediated release of GSK3β (step 3) from the degradation complex can simply be reversed by sufficiently high concentrations of GSK3β (step 6). In this case, however, the effect is small. Thus, axin blocks the action of Dsh so effectively that it renders the Dsh pathway inoperable. Figure 4 The Effect of Dsh versus Axin or GSK3β on the Half-Life of β-Catenin in Xenopus Extracts (A and B) Predicted effects of Dsh, axin, and GSK3β on the half-life of β-catenin degradation. The half-lives are calculated from simulated degradation curves. Data are plotted as function of added Dsh (logarithmic scale) for various concentrations of axin (A) and GSK3β (B). (C and D) Measured effects of Dsh, axin, and GSK3β on the half-life of β-catenin degradation. Stimulation of β-catenin degradation by axin occurs throughout the range of Dsh concentrations tested. (C) Axin increases the rate of β-catenin degradation even in the absence of added Dsh. (D) Stimulation of β-catenin degradation by GSK3β is detected only at high concentrations of Dsh. No effects of GSK3β on β-catenin degradation can be detected at less than 30 nM added Dsh. There is a disparity between the concentrations of axin in the experimental and theoretical curves. We assume that this is most likely due to the specific activity of the expressed axin protein. In Figure 4 C and 4D, we studied experimentally the dose-dependent effects of Dsh, GSK3β, and axin on β-catenin degradation. These curves represent β-catenin half-lives for various concentrations of axin ( Figure 4 C) and GSK3β ( Figure 4 D) with varying concentrations of Dsh. The results are qualitatively similar to those predicted by the model. As expected, β-catenin degradation is inhibited by increasing Dsh concentration and stimulated by increasing the concentration of either axin or GSK3β. There are, however, two pronounced differences in the effects of axin and GSK3β on Dsh inhibition. Whereas axin activates β-catenin degradation over a wide range of Dsh concentrations ( Figure 4 C), the effect of GSK3β becomes significant only at high concentrations of Dsh ( Figure 4 D). Furthermore, the inhibitory effect of Dsh can be almost completely blocked by high concentrations of axin (10 nM). In contrast, GSK3β (1 μM) can only partially inhibit the strong inhibitory effect of Dsh on β-catenin degradation. Our experimental results, however, show a smaller effect on the half-life of β-catenin degradation at high concentrations of Dsh as GSK3β levels are increased. Also, the concentrations of added axin in the theoretical curve and the experimental curves are very different. The quantitative difference between the model and experimental may simply reflect the fact that the specific activity of our GSK3β and axin preparations (purified from Sf9 cells and bacteria, respectively) may be low and that a significant fraction of the recombinant proteins may not be active. Alternatively, the low activity of GSK3β may point to an unidentified inhibitory activity present in our Xenopus egg extracts. Effects of Dynamic Changes in Protein Concentrations The dependence of flux on the concentration of a pathway component is a measure of how much the flux is sensitively controlled by that component. In metabolic control theory, the normalized concentration-dependent parameters of the total flux known as control coefficients have been very useful in defining the characteristics of pathways ( Heinrich and Rapoport 1974 ; Fell 1997 ). Similarly, in the analysis of bacterial chemotaxis, the response of a behavioral parameter as a function of changes in specific kinetic rates has been termed robustness ( Alon et al. 1999 ). Such terms are rarely measured in signal transduction. To determine the effects of changes in the levels of Wnt pathway components, we analyzed how the flux (β-catenin degradation) changes with changes in the concentrations of APC 0 , GSK3β 0 , Dsh 0 , and TCF 0 (see Figure S2 ). We chose to focus on the effects of changes in the concentrations of pathway components in the reference state, because similar effects were also seen for the stimulated state. Recently, we investigated a new and important property of the Wnt pathway, namely that the degradation of axin (reaction 15) is dependent on APC (unpublished data). The degradation rate of axin is mathematically expressed in the following manner: where K M represents a half-saturation constant for the activating effect of APC. The theoretical effect of APC on the concentrations of both β-catenin and axin is shown in Figure 5 , where we considered independently the effect of APC-mediated degradation of axin (“with regulatory loop” where Equation [5] is applied) or the absence of such an effect (where the linear rate equation ν 15 = k 15 axin is applied). With APC-mediated axin degradation, β-catenin degradation is affected very little by changes in the concentration of APC (25% decrease with a 2-fold increase in APC concentration). This resistance to changes of β-catenin levels upon changes in APC concentration is due to the APC-dependence of axin degradation (see Figure 1 and Equation [5]). Decreasing the concentration of APC inhibits the degradation of axin, thereby promoting the formation of the degradation complex. As shown in Figure 5 , in the absence of the regulatory loop, axin degradation is APC independent, homeostasis is lost, and β-catenin levels are greatly upregulated with decreasing APC concentrations. A comparison of the curves that represent the dependence and independence of axin degradation on APC (dashed lines in Figure 5 ) indicates that the regulatory loop acts in such a way that the normally inhibitory effect on β-catenin degradation as a result of lowering the concentration of APC is counteracted by an increase in axin levels. Figure 5 Effect of the Regulatory Loop for Axin Degradation The case “with regulatory loop” takes into account that axin degradation is APC-dependent (black curves). Alternatively, the case without this regulatory loop is considered (red curves). For the regulatory loop, the rate law (5) is used assuming that in the reference state the APC activation is half of its maximum ( K M = 98.0 nM). The value of k ′ 15 was chosen such that in the reference state both cases, with and without regulatory loop, yield the same degradation rate of axin ( k ′ 15 = 0.33 min −1 ). We have also simulated the effects of changes in the rate of β-catenin ( ν 12 ) and axin ( ν 14 ) synthesis on both β-catenin and axin levels (see Figure S3 ). Interestingly, changing the level of axin or β-catenin affects the concentration of the other component in different ways. An increase in the synthesis of axin results in a decrease in β-catenin, whereas increasing β-catenin synthesis leads to an increase in axin levels. This latter effect contrasts with effects observed upon changes of other parameters (see Figure S2 ) that affect the concentrations of axin and β-catenin in opposite directions. Transient Stimulation of the Pathway Wnt stimulation in vivo is transient, likely due to receptor inactivation/internalization and/or other downregulatory processes. We model transient Wnt stimulation by an exponential decay: where the reciprocal of λ represents the characteristic lifetime τ W of receptor stimulation and t 0 denotes the onset of signaling. The concentration changes of all other pathway compounds resulting from Wnt stimulation can be calculated by numerical solution of the system equations (see Dataset S1 ), with initial values of the variables corresponding to the reference state. Regulating axin turnover is important for Wnt signaling. Wnt-stimulated axin turnover has been reported in cultured mammalian cells ( Yamamoto et al. 1999 ) and in Drosophila ( Tolwinski et al. 2003 ). In a future paper we will show that axin turnover is affected inversely to β-catenin turnover by phosphorylation by GSK3β. Here we show theoretically that this regulated axin turnover sharply affects the dynamics of the response. Figure 6 shows the time-dependent behavior of the total concentration of β-catenin and the total concentration of axin upon transient Wnt stimulation. The concentration of β-catenin increases transiently and then returns to its initial value. In contrast, the concentration of axin is temporarily downregulated. Further analysis of Figure 6 reveals that the amplitude of the β-catenin signal upon transient stimulation is significantly lower than the steady-state concentration upon permanent stimulation ( W = 1; see Figure S1 ). The curves a and a′ in Figure 6 are calculated for the reference values of the rate of axin synthesis and of the rate constant of axin degradation, whereas the curves b and b′ and the curves c and c′ are obtained for the case where both parameters are increased by a factor of 5 and decreased by a factor of 5, respectively. Under these conditions, both the degradation rate and the synthesis rate are altered by the same factor, thus maintaining essentially identical steady-state concentrations of axin. As a result, the steady-state concentrations of axin are the same in the unstimulated condition ( W = 0) and after diminution of the Wnt signal; however, during active signaling, the differences in the dynamic nature of signal output at differing rates of axin turnover are dramatically revealed. Figure 6 Timecourse of β-Catenin and Axin Concentrations Following a Transient Wnt Stimulation Transient activation of the pathway is modeled assuming a Wnt stimulus that decays exponentially (Equation [6] with τ W = 1/ λ = 20 min) starting at t 0 = 0. The straight line for t < 0 corresponds to the steady state before pathway stimulation. The curves are obtained by numerical integration of the differential equation system (see Dataset S1). The various curves for β-catenin and for axin differ in the turnover rate of axin determined by the parameters ν 14 and k 15 (curves a: reference values of these parameters; curves b: increase by a factor of 5; curves c: reduction by a factor of 5). All other parameters are given in the legend of Table 2 . Interestingly, an increase in the turnover rate of axin leads to higher amplitudes and shorter durations of the β-catenin signal. This can be explained by the faster degradation of axin after its Dsh-mediated release from the destruction complex. Thus, β-catenin degradation is effectively inhibited for a certain time period due to a reduced availability of the scaffold axin. Since the steady-state concentration of free axin remains unchanged (rate of axin synthesis equals the rate of its degradation) during the transition from W = 0 to W = 1, a fast axin turnover favors rapid replenishment of the axin pool after the decline of the Wnt stimulus and, in this way, fast recovery of the destruction complex. This explains why the β-catenin signal is not only amplified, but becomes more spike-like. Increasing the turnover rate of axin affects the response of axin to temporary Wnt stimulation in a similar way as the response of β-catenin; i.e., the signal is amplified and sharpened ( Figure 6 ). Closer inspection of Figure 6 reveals that the axin response precedes the β-catenin response. For example, in the reference case, the β-catenin concentration reaches its maximum at about 260 min (curve a), whereas the minimum of the axin concentration is reached at 130 min (curve a′). This effect can be understood by observing that it is the lowering of the axin concentration that decreases the concentration of the destruction core complexes; in turn, this stabilizes β-catenin. Mechanistic Differences between APC and Axin as Scaffolds As the axin concentration is several orders of magnitude lower than that of the other components in the degradation pathway (see Table 1 ), we decided to test the effect of increasing axin levels (up to, equal to, and greater than the concentrations of other components in the pathway). To do this, we had to extend the model to include additional reactions, marked in blue in Figure 1 ; these had previously been neglected due to the very low axin concentrations. High axin concentrations affect most prominently the formation of the β-catenin/axin complex. Assuming a realistic value for the β-catenin–axin dissociation constant ( K 18 = 1 nM), a moderate increase in axin concentration should theoretically accelerate β-catenin degradation, whereas a much higher concentration should result in inhibition of β-catenin degradation, due to the formation of partial complexes on axin. A more extensive analysis of β-catenin half-lives over a range of axin concentrations shows such a biphasic curve ( Figure 7 A, curve b). These effects can also be seen experimentally in extracts ( Figure 7 B), where 10 nM axin accelerates and 300 nM axin inhibits β-catenin degradation. The t ½ decrease for low amounts of added axin can be easily explained by the fact that greater amounts of APC and GSK3β can be recruited to form the destruction complex. As a result, the t ½ decreases from 60 min to t ½ = 3 − 4 min. The inhibitory effect of axin becomes apparent only for axin concentrations approaching that of the other components. As shown in Figure 7 A, the effect of axin binding only to GSK3β ( K 19 = 1 nM, K 18 → ∞) only becomes inhibitory at higher than micromolar concentration (curve c), whereas the combined effect of binding to both β-catenin and GSK3β ( K 18 = 1 nM, K 19 = 1 nM) shows inhibition at less than 500 nM (curve d). If, however, we model an ordered process of binding to axin, then abortive inhibitory complexes cannot form. We show this in Figure 7 A. Here there is no separate binding of axin to β-catenin or GSK3β. In this case, there is no increase in the t ½ at high axin concentrations ( Figure 7 A, curve a). Figure 7 Effects of Increasing Axin Concentration on β-Catenin Degradation (A) Effect of axin concentration on β-catenin half-life. Curve a: reference case ( K 18 , K 19 > 1 nM, ordered mechanism); curve b: K 18 = 1 nM, K 19 > 1 nM; curve c: K 18 > 1 nM, K 19 = 1 nM; curve d: K 18 = 1 nM. (B) High concentration of axin inhibits β-catenin degradation in Xenopus egg extracts. Labeled β-catenin was incubated in Xenopus extracts in the absence (0 nM) or presence of moderate (10 nM) and high (300 nM) concentrations of axin. Moderate concentrations of axin greatly accelerate, whereas high concentrations inhibit β-catenin degradation. We also examined theoretically the effects of increasing APC concentration on the half-life of β-catenin, as shown in Figure 8 . The black curve corresponds to a nonordered mechanism, such as that found in axin, in which the β-catenin–APC dissociation constant (reaction 17) is low. The inhibitory effect of APC at high concentrations is due to its β-catenin buffering activity. The green curve corresponds to an ordered mechanism and reflects a high β-catenin–APC dissociation constant (high K 17 ). In this case, increasing concentrations of APC greater than the reference concentrations does not lead to inhibition of β-catenin degradation even at very high concentrations of APC. In cultured cells, overexpression of APC stimulates β-catenin degradation ( Munemitsu et al. 1995 ; Papkoff et al. 1996 ). Unfortunately, we are presently unable to express full-length APC in Xenopus egg extracts to measure the effects of high levels in the extract system. Figure 8 Effects of APC Concentrations on β-Catenin Degradation Effect of APC concentration on β-catenin half-life assuming an ordered (curve a) or nonordered mechanism (curve b: K 17 = 1,200 nM), respectively. β-Catenin can also be degraded by nonaxin-dependent mechanisms, which include Siah-1 and presenilin-mediated degradation. Though they are expected to contribute very little to the total flux through the pathway, the nonaxin-dependent processes may have very important influences under certain conditions. In our Xenopus system, these alternative pathways do not contribute greatly to the half-life of β-catenin. Experimentally, we have measured only a 1.5% contribution to total β-catenin degradation such that the half-life of β-catenin is 45 h when the axin-dependent processes are inhibited. If in some situations the nonaxin-dependent degradation contributed 10% to the flux, the half-life would be 6.3 h ( k 13 = 1.83 · 10 −3 min −1 ). The alternative pathways have very little effect on the half-life of β-catenin at normal and supranormal concentrations of APC. However, the effect of these alternative pathways becomes much more prominent when the APC concentration is lowered, a situation that may be significant under pathological conditions. As seen in Figure 9 A, when APC levels are at 50% of their normal concentration, there are dramatic differences in β-catenin concentration, depending on whether the alternative degradation pathway contributes to 1.5% or 10% of the total β-catenin degradation activity. The importance of the regulatory loop involving APC-mediated axin degradation is shown in Figure 9 B. In the absence of the regulatory loop, a significant inhibition of APC levels would strongly inhibit axin degradation, leading to a large increase in β-catenin levels. The control of β-catenin would be very brittle in this circumstance. However, by making axin degradation dependent on APC, a loss of APC would not stabilize axin levels, and the high axin levels would support continued degradation of β-catenin. This is the situation labeled “with regulatory loop” shown in Figure 9 B. The control of axin degradation could be a decisive factor in the response of the system to genetic or environmental effects on APC. Figure 9 Effects of the Alternative β-Catenin Degradation Pathway and of Axin Degradation at Low Concentrations of APC (A) The alternative β-catenin degradation pathway (axin independent) can have profound effects on β-catenin levels at low APC concentrations. Variations of β-catenin and axin resulting from changes in APC concentration were calculated from the standard stimulated state. Relative variations were plotted since variation in the share of alternative degradation (1%, 5%, and 10%) results in changes of the standard stimulated state (all parameters are constant). β-Catenin and axin levels for varying contributions of the alternative degradation pathway are as follows: 1.5%, β-catenin 178 nM, axin 0.00728 nM; 5%, β-catenin 151 nM, axin 0.00679 nM; 10%, β-catenin 125 nM, axin 0.00629 nM. (B) Inhibition of axin degradation reduces β-catenin concentration after loss of APC. Plotted is the concentration of a potential proteasome inhibitor I (scaled to its inhibition constant, K I ) necessary to reduce β-catenin concentration to its original level, depending on the concentration of APC. Control, Modular Composition, and Robustness of the Wnt Pathway The model contains many parameters that affect the system behavior in different ways and to various extents. We can systematically investigate these parameters and look for those whose perturbation the system is most sensitive or most robust against. We focus on the concentrations of β-catenin and axin and calculate the responses in the total concentrations of these two compounds upon changes in the rates of the individual processes. For quantifying the effects of the rate constants k +i and k −i , we use control coefficients for the total concentration of β-catenin and corresponding definitions for the control coefficients C axin ±i for the total axin concentration. These coefficients, originally proposed for quantifying control in metabolic networks (for reviews, see Heinrich and Schuster 1996 ; Fell 1997 ), describe the relative changes of the concentrations of the given compounds to relative changes of the rate constants. The control coefficients for the reference state are listed in Table 3 . It should be remembered that the following discussion refers to small perturbations of the reference state. Table 3 Control Coefficients for the Total Concentrations of β-Catenin and Axin and Parameters Quantifying the Sensitivity and the Robustness of the Wnt/β-Catenin Pathway The control coefficients (Equation 7) were obtained by numerical determination of the response to a change of the rate constants of all steps by 1%. Using relative changes of rate constants less than 1% does not lead to a significant improvement of the precision of the C values. Coefficients are given for the reference state. Horizontal lines separate the coefficients for distinct modules of the pathway. The last block contains the coefficients for parameters that enter the systems equations as binding rate constants k +j and dissociation rate constants k −j via dissociation constants K j = k −j / k +j . The upper signs of these coefficients refer to changes in k +j and the lower sign to changes in k −j . The sum of the control coefficients in each column is zero. Additional summation rules hold true for the rate constants within each module as well as for the two rate constants of each binding equilibrium. The standard deviation σ of the concentration control coefficients and the robustness ρ for β-catenin and axin are calculated by applying Equations (8) and (9) For the reference state, there are six steps exerting strong negative control on the total β-catenin concentration ( C βcat i ≅ −1). This group includes the reactions participating in assembling the destruction complex APC/axin/GSK3β. The corresponding parameters involve the rate constants k 7 for the binding of axin to APC, k 6 for the association of GSK3β to the APC/axin complex, and k 4 for the phosphosphorylation of axin and APC in the destruction complex. Similar strong negative control is exerted by β-catenin binding to the phosphorylated destruction complex (rate constant: k 8 ), the phosphorylation of β-catenin in the destruction complex (rate constant: k 9 ), and the synthesis of axin ( ν 14 ). Six other reactions exert strong positive control in the reference state on the total concentration of β-catenin (concentration ( C βcat i ≅ 1). To this group belong the reactions participating in the disassembly of the destruction complex APC/axin*/GSK3β, which are described by the rate constants k − 7 for the dissociation of the APC/axin complex, k − 6 for the dissociation of GSK3β from the destruction complex, and k 5 for the dephosphorylation of the APC and axin in the destruction complex. Other steps with a high positive control are the dissociation of β-catenin from the destruction complex (rate constant: k − 8 ), axin degradation (rate constant: k 15 ), and β-catenin synthesis ( ν 12 ). There are many reactions exerting almost no control on β-catenin levels in the reference state. This group includes binding of β-catenin to TCF and APC ( k 16 and k 17 ), and the corresponding dissociation processes ( k − 16 and k − 17 ; again only valid for small perturbations). Interestingly, the effects of the two β-catenin degradation processes (rate constants: k 11 and k 13 ) are also small. Calculation of control coefficients for the standard stimulated state reveals that some steps that exert no control in the reference state become important. These are the activation and inactivation of Dsh (rate constants: k 1 and k 2 ) and, more pronounced, the Dsh-mediated release of GSK3β from the destruction complex ( k 3 ). For all other processes, the signs of the control coefficients for β-catenin and axin do not change at the transition from the reference state to the standard stimulated state. The effects of parameter changes on axin are generally opposite to those on β-catenin; i.e., processes with a positive control coefficient for β-catenin have negative control coefficients for axin and vice versa. A significant exception is the synthesis of β-catenin, which exerts a positive control not only on β-catenin but also on axin, as expected from the results obtained in the last section. Closer inspection of Table 3 reveals that the values of the control coefficients for the rate constants sum up to zero. This fact is known as the summation theorem for concentration control ( Heinrich and Rapoport 1974 ) and is valid for all reaction networks at steady state. This result finds its explanation in the invariance of the steady-state concentrations against simultaneous change of all rate constants by the same factor. Interestingly, in the present case there are subgroups of processes whose control coefficients separately sum up to zero, indicating a modular structure of the pathway. In Table 3 , the control coefficients of the different modules are separated by horizontal lines. The main four subgroups are the Dsh module (not shown in Table 3 ), the kinase/phosphatase module, the β-catenin module, and the axin module. A subgroup is defined by a set of reactions where the control coefficients of the binding reactions are opposite to those of the corresponding dissociation reactions ( C +i = − C −i for i = 6, 7, 8, 16, 17). For those more familiar with genetic manipulation, it is more common to vary the concentrations of individual components rather than vary the rate constant of a reaction. Table 4 shows the control coefficients for β-catenin and axin calculated for changes in the total concentrations of pathway components instead of the rate constants. Using the values of Table 4 , the potential tumor-supressing effects (of APC, GSK3β, and axin) and potential oncogenic effects (of PP2A, TCF, Dsh, β-catenin) can be explained and quantified. It may be worth mentioning that there is no summation theorem for the control coefficients when calculated by changing total concentrations instead of rate constants. For practical reasons, it may be easier to discuss the coefficients with respect to concentration changes ( Table 4 ); for theoretical reasons, changing rate constants are simpler to handle. We think that eventually it will also be clearest to speak about oncogenic reactions instead of oncogenic genes, especially if we are thinking of oncogenesis in response to pharmacologic or environmental perturbations. Genetic defects then can be considered in terms of changes in activity, transcription, translation, or proteolysis. Table 4 Concentration Control Coefficients for the Total Concentrations of β-Catenin and Axin Relative to Changes in the Concentrations of Pathway Components The control coefficients were obtained by numerical determination of the response to a change of total concentrations by 1%. Coefficients are given for the reference state and for the standard stimulated state Clearly, the robustness of a variable towards parameter changes is higher the lower the corresponding concentration control coefficient. To arrive at an estimation of the overall effects of parameter perturbations on the system as a whole, we consider first the standard deviation σ of the control coefficients from their mean value. According to the summation theorem, the mean value of all control coefficients for a given variable is zero. Thus, we get for the standard deviation for the control coefficients of β-catenin: where the summation is performed over all reactions, including forward and backward steps of fast equilibria. High values of σ indicate that the given variable is on average very sensitive towards changes of rate constants. We propose to introduce a measure for the robustness ρ of a variable towards changes of all parameters in the following way: As σ may vary between zero and infinity, the range of ρ is confined to the interval 1≥ ρ ≥ 0. High values of ρ resulting from low σ values for the control coefficients indicate that the variable is robust against parameter perturbations. The standard deviations σ of the control coefficients and the ρ values for β-catenin and axin are presented in the last two rows of Table 3 . Because many control coefficients are close to zero and the absolute values of the others hardly exceed unity, the σ values for β-catenin as well as for axin are rather small. Since all values for σ are lower than unity, a 1% change in a rate constant leads, on average, to a response of <1% in the overall level of β-catenin. The total concentration of axin is more robust against parameter perturbations than the total concentration of β-catenin, particularly in the standard stimulated state. A transition from the reference to the standard stimulated state results in a lower robustness for β-catenin and a higher robustness for axin. Discussion Theory and quantitation are mutually dependent activities. It would seem unlikely that one would go to the trouble to measure detailed kinetic quantities without a specific model to test, and it is equally unlikely that realistic models can be constructed without the constraints of quantitative experimental data. Our intent in trying to reproduce a substantial part of the Wnt pathway in Xenopus egg extracts was to acquire the kind of detailed kinetic data required to build a realistic model. There are several unusual advantages to the extract system that contributed to this effort. The Xenopus egg extract is essentially neat cytoplasm; it reproduces the in vivo rate of β-catenin degradation and responds to known regulators as expected from in vivo experiments. Kinetic experiments with high time resolution are possible in this system, since a well-stirred extract is presumably synchronous in ways in which collections of cells may not be. In extracts it is possible to precisely set the level of components by depletion or addition. The direct output of the canonical Wnt pathway is an easily measured cytoplasmic event, the degradation of β-catenin. Thus, in this unusual system it is possible to acquire quantitative information about signaling pathways, not achievable in vivo. At the same time, these extracts have limitations. We have not considered the receptor events, and it is likely that reactions at the plasma membrane contribute to dynamic features. Also, our analysis is incomplete, as there are other components of Wnt signaling, such as casein kinase I δ , casein kinase I ɛ , and PAR1, as well as cross-talk from other pathways, that influence the behavior of the system. We have also oversimplified the multiple phosphorylation steps. We have assumed a simple interconversion of the phosphorylated and unphosphorylated complex of axin, APC, and GSK3β, whereas in reality multiple phosphorylation states exist within the complex; the states may be random or sequential. We simply do not have the information to provide a much more specific model of phosphorylation interconversions at this time, although the model could easily be extended. Finally, there is the question of what Wnt process we are studying. We are looking at events in the cytoplasm of unfertilized eggs. Though endowed with all of the core components of the Wnt pathway, the egg is, as far as we know, transcriptionally silent and not involved in Wnt signaling, though this system is active very soon in embryogenesis. Thus, there is no biological in vivo behavior with which to compare the in vitro behavior. Nevertheless, the basic core circuitry is intact and is presumably prepared for the early Wnt events in the embryo. All the properties of the egg extract system are very similar to that circuitry in vertebrate somatic cells. To build a mathematical description of the Wnt signaling system, we started with the basic circuitry discerned from previous studies in Xenopus embryos and mammalian cells, whose similarity to the in vitro system we had already confirmed. We derived a system of differential equations that described the time-dependent variations of the system variables, i.e., the concentrations of the pathway components and their complexes. Parameters of the model are binding constants of proteins, rate constants of phosphorylations and dephosphorylations, rate constants of protein degradation, and rates of protein synthesis. Model reduction was achieved by considering conservation relations and by applying rapid equilibrium approximations for selected binding processes. Despite these simplifications, the model consists of a nonlinear system of differential equations whose solution requires the use of computers. Not all of these parameters were accessible to measurement. To circumvent this problem, we used as primary inputs not only kinetic parameters characterizing individual steps, but quantities that are more easily accessible from experiment, such as the overall flux of β-catenin degradation. This allowed us to derive rate constants as well as protein concentrations in a reference state, where there was no Wnt signal. This state serves as a starting point for predicting the system behavior during Wnt signaling as well as after experimental perturbations. The basic model reproduced quantitatively the behavior of the reference state, including perturbations of this state achieved by varying the concentration of axin, GSK3β, and TCF. It also reproduced extensions of this to the signaling state. A wide variety of different sets of experimental data could be simulated by the same model, employing the same sets of kinetic parameters. We approached this process iteratively. For example, the early model did not include nonaxin-dependent degradation of β-catenin, but inclusion of this process improved the fit to the experimental data. More significantly, addition of this process had interesting biological implications, which we discuss. In many ways, one of the most peculiar findings was the very low concentration of axin in the Xenopus extracts. Axin levels in other organisms may similarly be very low: Drosophila axin can be detected by Western blotting only following its immunoprecipitation ( Willert et al. 1999 ). Although our theoretical and experimental studies have shown that axin is inhibitory at high concentrations, both indicate that axin is not present at the optimal concentration for the highest rate of β-catenin degradation. Therefore, axin levels are not set for optimality of β-catenin degradation, but are presumably optimized for some other purpose. Theoretically, axin levels must be held below the very sharp threshold of Dsh inhibition. Experimentally, these thresholds, which blunt Wnt signaling, are observed but are not as sharp as expected, and this may indicate some other compensatory effects. These thresholds would limit axin concentration to well below 1 nM if activated Dsh were constrained to concentrations of below 1 μM. Under these circumstances, we can expect that axin would never be found at concentrations approaching those of other Wnt pathway components (50–100 nM). The low concentration of axin relative to other components (such as GSK3β, Dsh, and APC) has another design feature potentially very general and important for the modularity of metazoan signaling pathways. Axin is a critical node point for controlling β-catenin levels, but it also interacts with components shared with several other important pathways. The interaction of these components with axin fluctuates due to Wnt signals (reflecting changes in binding as well as changes in axin levels), yet because the concentration of axin is so low, there will be no appreciable change in the overall levels of GSK3β, Dsh, or APC (all these components important in other pathways would otherwise be driven to fluctuate). The very low axin concentration thus isolates the Wnt pathway from perturbing other systems, a simple mechanism to achieve modularity. Other scaffold proteins may serve similar functions in other pathways. These insights follow from a very simple measurement of axin concentration and suggest the utility of measuring the levels of signaling pathway components in different cell types and circumstances. Since quantitative and kinetic features may be important in defining modules, it suggests that qualitative circuit diagrams of signal transduction may overlook very important design features. Modularity within the Wnt pathway can be defined by an extension of the summation theorem of Heinrich and Rapoport (1974 ), which argues that the steady state of an entire pathway would have control coefficients that added to zero. When the Wnt pathway is broken down to several subpathways, we find that within these subpathways the control coefficients would sum to zero at steady state. While some of this subdivision is obvious (i.e., the kinase phosphatase module involving the phosporylation of APC and axin complexed to GSK3β), in other cases, such as the β-catenin module, it is much less obvious. Here the reactions include the phosphorylation of β-catenin in the APC/axin/GSK3β complex, the release and degradation of β-catenin, and the synthesis and nonaxin-dependent degradation of β-catenin. Balanced perturbation of these subpathways as a whole will not affect the overall flux of β-catenin degradation. It is not clear whether this concept of modularity might be extended usefully in two other directions: modularity in systems not at steady state, i.e., with transients, and estimates of linkage between pathways by some definition of nonzero summations expressing the degree of independence or modularity. In addition to work by Kholodenko et al. (1997 ), this paper marks one of the first extensions of metabolic control theory to signal transduction. Metabolism and signal transduction seem very different, the former involving the transfer of mass and the latter the transfer of information. In addition, metabolic pathways generally involve dedicated components and the specificity of interaction of substrates and enzymes is very high. Signaling pathways share many components; interactions are often weak. Metabolism, which has had a long history of quantitative study, was a natural field for the development of control theory, and this theory has been successful in converting the specific information about the behavior of enzymes in a pathway to the overall behavior of metabolic circuits. Control coefficients are useful measures of the impact of a process or quantity on another. In its application to metabolism, it allowed us to dispose of erroneous concepts, such as the notion of a rate-determining step. In signal transduction, control coefficients might play a similar role. Here they can be used to indicate quantitively the effects of a particular reaction on some other property, such as flux through the pathway or concentration of another component. By this definition, certain rate constants, such as the phosphorylation and dephosphorylation of APC and axin, have a major influence on the levels of β-catenin, while others, such as the degradation rate of phosphorylated β-catenin, have little effect. The sign and magnitude of these control coefficients give some indication what gene products could be oncogenes or tumor suppressors. As shown in Table 4 , by this criterion APC, GSK3β, and axin are potent tumor suppressors, whereas β-catenin is an oncogene. Dsh would be expected to exert only moderate oncogenic effects. Clearly the effects of certain gene products are dependent on context, including their rate of synthesis and steady-state concentration. As our understanding of pathways improve, the effect of mutation or pharmacologic inhibition could be estimated quantitatively using control coefficients. The differences between cell types and organisms could be exploited to better predict mutagenic and pharmacologic impact on signal propagation. Despite considerable progress in identifying components of the Wnt pathway, many important mechanistic details are still lacking. In this analysis we have shown that Dsh seems to act to prevent the phosphorylation of the axin/APC complex, not the phosphorylation of β-catenin. Dsh (complexed to GBP) does not seem to be a general GSK3β inhibitor, like Li + , but rather is focused on the two scaffolding proteins. This was apparent from the biphasic nature of both the theoretical and experimental curves, which suggested that Dsh inhibited the rephosphorylation of axin/APC, but still allowed many cycles of β-catenin phosphorylation, ubiquitination, and degradation. This mechanism was further proven by a timing-of-addition experiment. It needs to be further confirmed and extended by looking specifically at individual phosphorylation sites on all the components of the complex. Another insight into the mechanisms of complex formation and control of β-catenin degradation concerns the inhibition of β-catenin degradation at concentrations of axin approaching those of other components. This suggests that axin binds APC, GSK3β, and β-catenin in random order. As discussed above, the axin concentration is limited by other factors; owing to the low concentration of axin, random binding is not likely ever to be a problem. The situation for APC seems very different. The concentration of APC is comparable to that of the other components. Overexpression studies show no inhibitory effects. These theoretical and experimental observations suggest that APC as a scaffold must be very different from axin as a scaffold. Most likely, APC binds components in an ordered manner. Metabolic pathways are understandable in terms of the familiar logic of chemical synthesis; signal transduction pathways, by contrast, often do not seem to conform to simple design principles. It is not clear at all whether signaling pathways have been optimized for a specific function or instead whether they are remnants of some early and rather arbitrary evolutionary experiment, now embedded in other processes that are difficult to change. Systems analysis, along with experiment, offers some hope of uncovering latent principles of design. For example, the modeling of the Wnt pathway gave a theoretical insight into the function of axin degradation in Wnt signaling. The degree of axin instability dramatically affects the amplitude and duration of the β-catenin response to a transient Wnt signal. If axin turnover were designed to be slow, then β-catenin would rise slowly to a low amplitude and persist for many hours. In a system where axin turnover was more rapid, the amplitude could increase several-fold and would persist a shorter time. The duration and amplitude of the response are likely to be important factors in developmental systems, which may respond differently to different amplitudes and durations of a signal. In addition, some developmental processes occur with such rapidity that the same signal would be interpreted differently at different times, hence the need to quickly terminate a signal. The very different effects of transient and persistent signals in the same pathway have been studied in PC12 cells in the MAP kinase pathway activated by EGF or NGF ( Marshall 1995 ). Finally, APC-dependent axin degradation stabilizes the Wnt pathway to variations in the APC concentration. Viewed from this perspective, the regulatory loop involving APC and axin degradation is an important design feature of the Wnt pathway. Robustness has also been considered an important design principle for signaling processes ( Alon et al. 1999 ), and control coefficients can be a good measure of this robustness. We present here a general measure of robustness of the entire pathway, using a measure that sums the variation in every individual reaction. Though it is generally thought that “robust” is good, the complement of robust is adaptability, and it may be that some aspects of a signaling pathway are designed to be responsive to changes in some parameters so that the same pathway can be used differently in different circumstances by altering key parameters. Since quantitative measures of the concentration or posttranslational modification of signaling proteins are rare in the literature, we have very little information on whether the organism varies certain key components to achieve different behavior of the signaling systems. Another aspect of robustness is susceptibility to mutation or pharmacologic or environmental perturbation. The unexpected minimal phenotypes observed in numerous mouse knockout experiments have underscored our ignorance of the adaptable responses of organisms and in particular the adaptable nature of signaling pathways. One unexpected theoretical observation in this paper was the potential importance of the nonaxin-dependent degradation for β-catenin, under conditions where the APC levels are reduced. In our model, the nonaxin-dependent degradation contributed only a few percent to the overall flux of β-catenin degradation. Yet if the APC levels fall only 50%, the exact level of the alternative pathway made a large difference in the steady-state level of β-catenin. If the activity of the alternative pathways varied in different tissues, then this simple but largely silent effect could explain the tissue specificity of APC mutations. Similarly, variations in the alternative pathway might also explain some aspect of individual risk to loss of a single copy of the APC gene. An experiment can better be judged by how many questions it raises than by how many it answers. The same may be said about theoretical analysis. Such analysis is always a work in progress, in that the experimental basis is continually changing to some degree. Some of the experimental changes, though significant mechanistically, may have little effect on the model and its interpretation. Some may require major revision. In the case of the Wnt pathway, the theoretical analysis and modeling have already raised several interesting questions of biological importance. They have already stimulated further experimentation. More than anything, the modeling has increased the urgency for obtaining accurate quantitative information about both steady-state and transient processes in Wnt signaling and for obtaining information about the differences in parameters in different tissues and in different organisms. Materials and Methods Egg extracts and degradation assays. Xenopus egg extracts were prepared as described previously ( Salic et al. 2000 ). Extracts were used either used immediately or stored at –80°C after being snap-frozen in liquid nitrogen. β-Catenin degradation assays were performed as described before ( Salic et al. 2000 ). Measurement of β-catenin synthesis. Freshly prepared Xenopus egg extracts were either supplemented with 25 mM LiCl in Xenopus buffer (XB) or XB and an aliquot were withdrawn for β-catenin degradation assays. The free methionine pool in Xenopus embryos is approximately 90 μM, and based on this number, [ 35 S]methionine was added to the extract to a give a final activity of 1,000 counts per picomole methionine. Extracts were incubated at 20°C, and aliquots were withdrawn at the indicated times for SDS-PAGE, trichloroacetic acid (TCA) precipitation, and β-catenin pull-downs. To assay protein synthesis in the extract, total methionine incorporation was measured by TCA precipitation. In brief, Xenopus extracts (2 μl), metabolically labeled with [ 35 S]methionine as above, were diluted to 100 μl with PBS and supplemented with 2 μl of 2% deoxycholate and TCA to 5%. The reaction was pelleted at 20,000 × g for 10 min at 4°C. After washing with ice-cold acetone and air drying, the radioactivity of the pellet was measured in a scintillation counter. To isolate metabolically labeled β-catenin from Xenopus extracts, we used His-tagged APCm3 cross-linked to Ultralink beads (Pierce, Woburn, Massachusetts, United States). Since phosphorylated APC has a much higher affinity for β-catenin, APCm3 beads were first phosphorylated with 300 nM His-tagged GSK3β in 25 mM HEPES (pH 7.7), 1 mM EDTA, 300 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, and 1 mM ATP for 1 h at room temperature with shaking. The beads were washed three times with 25 mM HEPES (pH 7.7), 1 mM EDTA, 300 mM NaCl, 1% Tween, and 1 mM DTT and were then used to pull down β-catenin. Extracts (50 μl) labeled with [ 35 S]methionine (see above) were diluted 5× with XB containing 1% Tween and protease inhibitors. The diluted extracts were incubated with phosphorylated APCm3 beads (20 μl) at 4°C for 2 h. The beads were washed and bound protein was eluted by boiling in SDS-PAGE loading buffer. Labeled β-catenin was detected following SDS-PAGE and autoradiography. Measuring dissociation rates of phosphorylated/nonphosphorylated β-catenin from axin. Radiolabeled β-catenin (5 μl) was phosphorylated using 300 nM His-tagged GSK3β and 100 nM maltose-binding protein (MBP)–axin in 20 μl of XB containing 10 mM ATP, 20 mM MgSO 4, and 50 mM NaCl. For the nonphorphorylated control, β-catenin was incubated as above, except that 50 mM LiCl was used instead of NaCl to inhibit GSK3β. The kinase reactions were incubated in a shaker for 30 min at 20°C and then added to 50 μl of MBP–axin, bound to amylose beads (1 mg of protein per milliliter of beads), and brought up to 250 μl with XB containing 50 mM LiCl. After phorsphorylated and nonphosphorylated β-catenin, respectively, bound to axin beads, the beads were washed three times with 500 μl of XB containing 50 mM LiCl. Dissociation of the bound β-catenin was initiated by adding 1 μM unlabeled recombinant β-catenin (His-tagged, from Sf9 cells) and incubated at 20°C in a shaker. At the appropriate times, 5 μl aliquots of beads were quickly removed, filtered through Wizard minicolumns (Promega, Madison, Wisconsin, United States), and washed with ice-cold XB (3 ml). Proteins bound to beads were eluted from the minicolumns with 20 μl of hot sample buffer, followed by SDS-PAGE and autoradiography. Recombinant proteins. The expression and purification of all recombinant proteins have been previously described ( Salic et al. 2000 ). Dsh was expressed as an MBP fusion in bacteria. His-tagged GSK3β, His-tagged Tcf3, His-tagged APCm3, and MBP–axin were expressed in Sf9 cells. Supporting Information Dataset S1 The Roles of APC and Axin Derived from Experimental and Theoretical Analysis of the Wnt Pathway (287 KB DOC). Click here for additional data file. Figure S1 Effect of Wnt Stimulation on the Concentrations of β-Catenin and Axin The curves represent steady-state concentrations of β-catenin (solid lines) and axin (broken lines) as functions of the strength W of Wnt stimulation. Curve a: free unphosphorylated β-catenin; curve b: free phosphorylated β-catenin; curve c: total β-catenin; curve d: total axin. All concentrations are scaled with respect to their values in the reference state. It is worth mentioning that in the model “without regulatory loop,” the steady-state concentration of free axin is determined by the condition X 12 = ν 14 /k 15 , and is, therefore, independent of Wnt stimulation. (2,954 KB TIFF). Click here for additional data file. Figure S2 Effects of the Amounts of Pathway Components on the Concentrations of β-Catenin and Axin This figure gives additional information with respect to the effects of Dsh, TCF, and GSK3β on the steady-state concentrations of total β-catenin (solid lines) and total axin (dashed lines) for the case of permanent Wnt-stimulation, W = 1. All concentrations and synthesis rates are scaled with respect to their values in the stimulated stationary state. (3,472 KB TIFF). Click here for additional data file. Figure S3 Effects of Synthesis Rates on the Concentrations of β-catenin and Axin The curves represent steady-state values of total concentrations of β-catenin (solid lines) and axin (dashed lines), depending on the rates of synthesis of β-catenin and axin. All concentrations and synthesis rates are scaled with respect to their values in the stimulated stationary state. (3,483 KB TIFF). Click here for additional data file. Table S1 Mathematical Notation for Model Variables as Subdivided into Independent and Dependent Variables (45 KB DOC). Click here for additional data file. Table S2 Complete List of Model Parameters of the Wnt Signal Transduction Model The rate constants marked with “#” play a role only in stimulated states where W ≠ 0. Note that some of the numerical values are given in a higher precision compared to Table 1 . (111 KB DOC). Click here for additional data file.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC212691.xml
544854	Transcriptional regulation of lipid metabolism by fatty acids: a key determinant of pancreatic β-cell function	Background Optimal pancreatic β-cell function is essential for the regulation of glucose homeostasis in both humans and animals and its impairment leads to the development of diabetes. Type 2 diabetes is a polygenic disease aggravated by environmental factors such as low physical activity or a hypercaloric high-fat diet. Results Free fatty acids represent an important factor linking excess fat mass to type 2 diabetes. Several studies have shown that chronically elevated free fatty acids have a negative effect on β-cell function leading to elevated insulin secretion basally but with an impaired response to glucose. The transcription factors PPARα, PPARγ and SREBP-1c respond to changing fat concentrations in tissues, thereby coordinating the genomic response to altered metabolic conditions to promote either fat storage or catabolism. These transcription factors have been identified in β-cells and it appears that each may exert influence on β-cell function in health and disease. Conclusion The role of the PPARs and SREBP-1c as potential mediators of lipotoxicity is an emerging area of interest.	Introduction Fatty acids are physiologically important both structurally, as components of phospholipids and glycolipids, as well as functionally, as fuel molecules. Metabolites of fatty acids, such as leukotrienes or prostaglandins, act as potent mediators in many biological processes. Fatty acids provide energy [ 1 , 2 ], particularly in the fasted state (Figure 1 ), but abnormalities in the metabolism of fatty acids can contribute to the pathogenesis of obesity and type 2 diabetes. Figure 1 Schematic diagram of fatty acid metabolism in the fasted state . Counter-regulatory hormones such as catecholamines act on adipocytes to increase lipolysis via hormone-sensitive lipase (HSL). Circulating FFA enter the cell and are converted to acyl CoAs, catalyzed by acyl CoA synthase (ACS). Acyl CoA enter the mitochondria via carnitine palmitoyl transferase-I (solid square) and enter the β-oxidation cycle (stippled circle) to produce acetyl CoA that is then available for further metabolism in the TCA cycle, leading to increased ATP and substrates for anaplerosis. In the β-cell, acyl CoA also participate as signalling molecules to promote insulin secretion (see text). Type 2 diabetes and free fatty acids Diabetes affects 6 % of the adult population and, with a growth rate of 6% per year, it is estimated that 200 to 300 million people worldwide will be afflicted by the end of this decade [ 3 ]. Type 1 diabetes, which accounts for < 10 % of all cases of diabetes [ 4 ], results from autoimmune-mediated destruction of pancreatic β-cells. The destruction may occur over months to years and can result in complete loss of the endogenous insulin supply and therefore results in exogenous insulin dependency. Type 2 diabetes, which accounts for 90 to 95 % of diabetes cases worldwide, is a heterogeneous disorder and its prevalence is rising. Type 2 diabetes is accompanied by chronic insulin resistance and a progressive decline in β-cell function [ 5 ]. Obesity is a major risk factor for the development of type 2 diabetes [ 6 ] and is believed to confer increased risk through obesity-associated insulin resistance [ 7 ]. Type 2 diabetes is often associated with hypertriglyceridemia or increased circulating concentrations of free fatty acids (FFA) [ 8 ]. Therefore, type 2 diabetes can be considered a lipid disorder as well as a disease of glucose intolerance [ 9 ]. Metabolism of fatty acids in the beta cell and insulin secretion Fatty acids, not glucose, are the major endogenous energy source for unstimulated islets [ 10 ]. This is consistent with the observation that although islets contain little glycogen, they maintain high rates of oxygen consumption in the absence of glucose [ 11 ]. Stimulation of islets by glucose diminishes fatty acid oxidation and increases total respiration [ 12 ]. Thus, rising post-prandial plasma glucose shifts the β-cells from fatty acids to glucose as an oxidative fuel. However, plasma concentrations of other nutrients such as FFA and amino acids can modulate the process of glucose-induced insulin secretion [ 9 ]. The plasma levels of nutrient metabolites vary with dietary composition. Thus, feeding behavior plays an important role in the control of islet β-cell function [ 13 , 14 ]. Short-term (2–6 hours) elevation of the plasma FFA concentration in human subjects [ 15 ] and animals [ 16 , 17 ] enhances while an acute decrease inhibits glucose-stimulated insulin secretion [ 15 , 18 ]. Following lipid infusion or ingestion of a mixed meal, the plasma FFA concentration rises and FFA diffuse into the β-cells [ 19 ]. Within the cytosol, fatty acids are converted to their fatty acyl CoA derivatives (Figure 1 ), which in turn augment insulin secretion via different signalling mechanisms: increased formation of phosphatidic acid and diacylglycerol, which directly and indirectly (through activation of protein kinase C) enhance exocytosis of insulin stored within secretory granules; stimulation of endoplasmic reticulum Ca 2+ -adenosine triphosphatase, leading to an increase in intracellular calcium concentration and augmentation of insulin secretion; and closure of the K + - ATP channel with resultant depolarization of the β-cell membrane, which causes an increase in intracellular Ca 2+ and stimulation of exocytosis of insulin-containing granules [ 21 ]. In addition to being oxidized, glucose can be metabolized through anaplerotic processes to increase malonyl CoA concentrations in the β-cell. Malonyl CoA inhibits CPT-I, thus impairing the transport of fatty acyl CoAs into the mitochondria where they would be oxidized [ 20 , 21 ]. The fact that de novo fatty acid synthesis in the β-cell is very low [ 22 ] indicates that malonyl-CoA is used as a switch compound, not as a precursor or effector molecule like long chain fatty acyl-CoA. The cytosolic concentration of long chain fatty acyl-CoA is controlled by feedback inhibition of acyl-CoA synthetase, and is buffered by fatty acid and long chain fatty acyl-CoA binding proteins [ 23 ]. The total concentration of long chain fatty acyl-CoA in livers of fed and fasted rats, is about 95 and 220 nmol/g dry weight, respectively [ 24 ], however quantification of cytosolic long chain fatty acyl-CoA in other tissues has yet to be done. In contrast to the acute effect of elevated plasma FFA to enhance insulin secretion, longer-term (> 48 h) exposure results in an impaired β-cell response to glucose both in vitro and in vivo in animals [ 25 , 26 ] and humans [ 27 - 31 ]. The inhibitory effect of chronically elevated plasma FFA is more prominent in individuals with a genetic predisposition to develop type 2 diabetes [ 32 ], thus a reduction in the plasma FFA concentration in type 2 diabetes improves insulin secretion [ 32 , 33 ]. The term lipotoxicity describes the deleterious effect of chronic FFA elevation on insulin secretion from the pancreatic β-cell [ 34 ]. In the Zucker diabetic fatty rat, chronically increased plasma FFA levels lead initially to a physiological impairment in insulin secretion. With time, β-cell mass is reduced by more than 50 % [ 26 ]. Within the β-cell, elevated fatty acyl CoAs increase the formation of ceramide, a sphingolipid. Ceramide, in turn, augments the formation of the inducible isoform of nitric oxide, which is toxic to the β-cell [ 35 ]. Incubation of human islets with FFA or ceramide has been shown to cause β-cell apoptosis [ 36 ]. Transcriptional regulation of free fatty acid metabolism Free fatty acid metabolism responds to varying metabolic states partially by induction of enzymes that promote either catabolic or anabolic processes. There are two major classes of transcriptional regulators of enzymes involved in fatty acid metabolism, the peroxisome proliferator-activated receptors (PPARs) and the sterol regulatory element binding proteins (SREBPs), which both exist in several isoforms. In general, PPARγ and SREBP-1c regulate processes involved in lipogenesis whereas lipolytic enzymes are induced by PPARα [ 37 ]. Peroxisome proliferator-activated receptors The PPARs form a subfamily in the nuclear receptor superfamily. PPARs, like other nuclear receptors, regulate gene expression in response to specific ligands through their actions as transcription factors. Peroxisomes contain PPAR-regulated enzymes involved in fatty acid β-oxidation [ 38 ]. Genetic deficiencies in peroxisome biogenesis in the human cause an accumulation of long chain fatty acids in cells [ 39 ]. So far, three isoforms encoded by separate genes and designated PPARα, PPARδ and PPARγ have been identified [ 40 ]. PPARα PPARα was the first member of this nuclear receptor subclass to be described. PPARα is expressed in numerous metabolically active tissues including liver, kidney, heart, skeletal muscle, brown fat [ 41 - 43 ], and also in monocytes [ 44 ], vascular endothelium [ 45 ] and vascular smooth muscle cells [ 46 ]. PPARα plays an important role in the regulation of cellular uptake, activation and β-oxidation of fatty acids. The natural, preferentially-binding ligands of PPARα are long chain unsaturated fatty acids including arachidonic acid, linoleic acid, and oleic acid but saturated fatty acids like palmitic acid can also act as ligands [ 47 ]. In hepatocytes and other tissues where it has been studied, ligand-activated PPARα binds to peroxisome proliferator response elements (PPRE) of DNA (Figure 2 ) and up-regulates transcription of genes involved in lipid catabolism and lipoprotein metabolism (Table 1 ) [ 48 , 49 ]. Consequently PPARα serves as a long chain fatty acid sensor that leads to autoregulation of long chain fatty acid metabolism mainly in the liver and heart and to a lesser extent in muscle, thus decreasing tissue content of lipids and minimizing lipotoxicity as circulating levels fluctuate [ 50 ]. Activation of PPARα also induces hepatic proliferation, hepatomegaly and hepatocarcinogensis in animal [ 51 ] but not human liver [ 52 ]. Obesity is a major risk factor in the development of type 2 diabetes and PPARα may affect body weight through regulation of fatty acid catabolism or expending energy [ 53 ]. PPARα ligands (such as fibrate drugs) could therefore improve insulin sensitivity by reducing lipid accumulation in tissues [ 54 ]. Figure 2 Overview of PPAR activation and effects . FFA (eg. oleic acid) interact with PPAR, which dimerize with retinoid X receptor (RXR) and translocate to the nucleus where the complex interacts with PPRE to activate gene transcription. The general effects of transcriptional activation of PPARα, PPARδ and PPARγ are shown on the right of the figure. Table 1 Selected hepatic PPARα regulated genes with at least one functional peroxisome proliferator receptor element (PPRE) identified within the promoter sequence Gene Function Species References Acyl CoA binding protein fatty acyl-CoA ester transport rat 127 Acyl CoA oxidase peroxisomal β-oxidation rat, human 128-130 Apolipoprotein-AI and AII plasma HDL metabolism human, mouse, rat 131-134 Apolipoprotein-AV plasma triglyceride metabolism human 134 Apolipoprotein-CIII plasma HDL metabolism rat 135 Bifunctional enzyme peroxisomal β-oxidation rat 136 Carnitine palmitoyl transferase-I and -II mitochondrial β-oxidation human, mouse, rat, hamster 132, 137-139 Cytochrome P450 enzymes fatty acid and cholesterol metabolism rat, mouse, human 130, 141-145 Δ6- and Δ5-desaturase desaturation of fatty acyl-CoA mouse 146 Fatty acid binding protein fatty acid binding/transport mouse 147 Fatty acid transport protein and translocase fatty acid transport mouse 148, 149 Lipoprotein lipase triglyceride clearance mouse 148, 149 Liver X receptor α cholesterol metabolism mouse 150, 151 Long-chain acyl-CoA synthetase fatty acid activation human, mouse 139, 152 Malic enzyme fatty acid synthesis mouse, rat 153, 154 Mitochondrial HMG-CoA synthase ketogenesis rat, human 152, 155 Medium-chain acyl-CoA dehydrogenase mitochondrial β-oxidation mouse 138, 139 Phospholipid transfer protein HDL metabolism human 156 Stearoyl-CoA desaturase-1 desaturation of fatty acyl CoA mouse 157 Superoxide dismutase free radical metabolism rat 158 Thiolase B mitochondrial β-oxidation rat 159 Transferrin iron transport human 160 Very long- and long-chain acyl CoA dehydrogenases mitochondrial β-oxidation mouse 139 Abbreviations: HDL, high density lipoprotein; HMG-CoA, hydroxymethylglutaryl-Coenzyme A PPARδ PPARδ was initially reported as PPARβ in Xenopus laevis [ 49 ]. Subsequently, the receptor was cloned in the human [ 55 ] as well as in rodents [ 56 ] and was named PPARδ. PPARδ is expressed in a wide range of tissues and cells with the highest levels of expression found in digestive tract, heart, kidney, liver, adipose and brain [ 57 ]. Saturated and unsaturated fatty acids are natural ligands for PPARδ [ 58 , 59 ]. PPARδ is implicated in adipocyte differentiation, which is induced by long-chain fatty acids [ 60 ]. In skeletal muscle, activation of PPARδ results in induction of proteins involved in lipid catabolism, cholesterol efflux and respiratory coupling in skeletal muscle independent from the effects of PPARα and PPARγ agonists [ 61 ]. PPARγ PPARγ stimulates fatty acid storage in adipose tissue by increasing both the storage capacity and the fatty acid flux into adipocytes. PPARγ is expressed in many cell types, including epithelial cells, B and T cells, macrophages, endothelial cells, smooth muscle cells [ 62 , 63 ] and predominantly in adipose tissue where it is necessary for the differentiation of adipocytes [ 64 ]. There are 2 splice variant of the isoform called PPARγ1 and γ2; the expression distribution of PPARγ2 is more limited than that of PPARγ1 [ 65 ]. The natural ligands of PPARγ are several unsaturated fatty acids such as oleate, linoleate, eicosapentaenoic and arachidonic acids [ 53 ]. Members of the thiazolidinedione (TZD) family, which are known as antidiabetic compounds, are synthetic ligands of PPARγ [ 54 ]. In adipocytes, PPARγ increases the expression of numerous genes involved in lipid metabolism and uptake [ 66 , 67 ]. Activation of PPARγ also induces adipocyte apoptosis, which is restricted primarily to large fully differentiated adipocytes [ 68 ]. This pro-apoptotic effect of PPARγ activation on large adipocytes, coupled with its capacity to enhance differentiation of adipocytes de novo , favours the formation of small adipocytes that tend to replace the large adipocytes normally constituting white adipose tissue [ 68 ]. PPARγ also negatively regulates transcription of several genes that impair insulin action, including tumor necrosis factor-α (TNFα) and leptin, proinflammatory cytokines produced by adipocytes and associated with insulin resistance [ 69 - 72 ]. Thus, the TZD drugs lower hyperglycemia, hyperinsulinemia and hypertriglyceridemia by indirectly enhancing the sensitivity of tissues to insulin, especially in skeletal muscle. However, the function of PPARγ is not restricted to adipogenesis and insulin sensitization. In peripheral monocytes and macrophages, PPARγ agonists inhibit the production of inflammatory cytokines [ 73 ] and induce differentiation and apoptosis in various cancer cells [ 74 , 75 ]. Peroxisome proliferator-activated receptors and β-cell function Both PPARα and PPARγ have been detected in pancreatic β-cells [ 76 , 77 ]. One caveat that complicates interpretation of some of the work described below is that PPARα and PPARγ agonists have effects on β-cell function independent of their interaction with the transcription factors. Thus, both fibrates and TZD can alter ATP-dependent K channel activity and rapidly (within 10 minutes) increase insulin secretion [ 78 ]. In addition, the generalized metabolic effects of these compounds may mean that effects observed in vitro on isolated islets may not apply to the in vivo situation. Therefore, the mode of delivery of the agents (directly onto islets versus in diets) and the time frame of study are important considerations. In pancreatic islets, exposure to long chain fatty acids (mixed unsaturated and saturated) induces PPARα expression [ 76 ] whereas high glucose in vitro or hyperglycemia in vivo suppresses expression [ 79 , 80 ]. Artificial ligands of PPARα such as WY14643 and clofibrate also induce PPARα expression in rat islets [ 76 , 81 , 82 ]. Similar to hepatocytes, this leads to up-regulation of enzymes favouring lipolysis, including acyl-CoA oxidase [ 76 , 81 ], pyruvate dehydrogenase-4 [ 82 ] and CPT-I [ 76 , 81 ]. The question arises as to the role of PPARα in the physiological regulation of insulin secretion. Its induction by long chain fatty acids and its ability to augment the insulin response to low glucose [ 81 ] suggests that it may play a role in sustaining β-cell secretory capacity during normal, cyclical periods of fasting. Thus, when glucose is low, PPARα will be induced, favouring β-oxidation of lipids to maintain β-cell ATP at a maintenance level. Moreover, the ability of β-cells to oxidize lipids is a critical for resumption of glucose-stimulated insulin secretion at the end of the fasting period [ 17 ]. However, when glucose is elevated above basal, PPARα will be reduced, allowing efficient glucose metabolism-dependent insulin secretion while inhibiting fatty acid oxidation. Overall, the effect of oscillating PPARα activity inversely with glucose concentration may help to maintain glucose responsiveness of the β-cell [ 83 ]. Four-to-six-hour fasted PPARα KO mice had normal circulating insulin [ 84 , 85 ] and their islets had normal glucose sensitivity [ 84 ] whereas 24 hour fasted mice had a 3-fold increase in circulating insulin [ 85 ]. The longer-term fast would allow for greater adaptation to occur; higher fasting insulin may reflect hepatic insulin resistance rather than altered β-cell function. In addition to these postulated mechanisms of PPARα control over β-cell glucose and lipid metabolism, it has also been proposed that amino acid metabolism might be affected. In the liver, an increase in PPARα is associated with a decrease in amino acid catabolism [ 86 ]. Because glutamine metabolites are potential signaling molecules in the β-cell [ 87 ], PPARα induction under conditions of low glucose could impair glucose-stimulated insulin secretion via its effects on glutamine catabolism [ 83 ]. This hypothesis has yet to be proven. In pathophysiological conditions involving deranged glucose and lipid metabolism, altered expression of PPARα may be important in the β-cell's lack of glucose responsiveness. In Zucker diabetic fatty rat islets, despite chronic hyperlipidemia, expression of PPARα, acyl-CoA oxidase and CPT-I mRNA is reduced [ 88 ]. It has thus been proposed that glucose is the dominant regulator of PPARα in the β-cell and that its suppression is a component of glucolipoxicity [ 89 ]. Glucolipotoxicity is a state in which β-cells are exposed to elevated plasma concentrations of both glucose and FFA, as is the case in insulin resistance. Several signalling pathways of the β-cell may be affected by altered PPARα expression and the overall outcome is predicted to depend upon whether fat or glucose has the dominant effect. In cases where glucose is elevated relative to lipid, a chronic reduction in PPARα would be expected to decrease the lipid oxidizing capacity of the β-cell [ 88 , 89 ], eliminating a detoxification route for fat metabolites [ 89 ]. Accumulation of lipids, for example as triglyceride within the β-cell, is associated with impaired glucose-stimulated insulin secretion, increased ceramide formation and apoptosis [ 88 ]. When lipid is chronically elevated relative to carbohydrate, induction of PPARα presumably would cause strong up-regulation of fat oxidizing genes but also UCP2 (see below), which would suppress glucose-stimulated insulin secretion. The implication of these hypotheses is that either too much or too little PPARα would impair β-cell function. Evidence in the literature supports this contention when in vitro models are employed. Notably, culture of islets or INS-1 cells with high glucose (6–20 mM) for 48 hours strongly suppresses PPARα protein expression by 80%. As predicted, fatty acid oxidation and glucose-stimulated insulin secretion are attenuated, while islet triglyceride and lipid esterification are increased [ 79 ]. Conversely, induction of endogenous β-cell PPARα (with clofibrate) leads to an increase in CPT-I expression and fatty acid oxidation, resulting in blunted basal and glucose-stimulated insulin secretion [ 90 ]. However, the situation is less clear when experiments are performed in vivo , leading to the conclusion that activation of PPARα in tissues other than β-cells causes indirect effects on insulin secretion secondary to changes in peripheral insulin sensitivity [ 83 ]. Thus, type 2 diabetic mice given dietary WY14,643, a PPARα agonist, have normalized serum lipids, glucose and insulin. PPARα activation also improves glucose-stimulated insulin secretion, reduces β-cell proliferation and β-cell mass compared with untreated controls [ 91 ]. Similarly, fenofibrate-treated obese diabetes-prone OLETF rats retain β-cell mass and have lower islet triglyceride content and fatty oxidation than untreated animals [ 92 ]. In both cases, the effects on β-cells are likely secondary to the observed weight loss and increase in insulin sensitivity of peripheral tissues. Chronic induction of PPARα may influence also insulin secretion indirectly because PPRE have been found in the promoter region of uncoupling protein-2 (UCP2) [ 93 ]. In general, uncoupling proteins (numbered 1–3 in order of their discovery) decrease metabolic efficiency by dissociating ATP synthesis from substrate oxidation in the mitochondrion by promoting translocation of protons from the inter-membrane space, across the inner mitochondrial membrane to the matrix [ 94 ]. Therefore, circumstances that limit mitochondrial proton gradient formation, such as up-regulation of UCP2 expression and activity, are predicted to limit insulin secretion. A study specifically examining the role of PPARα by use of the ligand clofibrate demonstrated induction UCP2 in islets [ 90 ]. In liver, stimulation of PPARα (or PPARδ when PPARα was absent) caused induction of UCP2 [ 95 ]. UCP2 expression inversely correlates with β-cell ATP and glucose-stimulated insulin secretion [ 96 - 99 ]. The significance of these findings is that up-regulation of UCP2 expression suppresses glucose-stimulated insulin secretion and is implicated as a potential contributor to lipotoxic effects mediated by PPARα in β-cells. PPARγ may also be an important transcriptional regulator of both normal and abnormal metabolism in pancreatic β-cells. In hyperglycemic, pancreatectomized rats the expression of PPARγ mRNA is increased [ 80 ] but others found that high fat but not high glucose up-regulates PPARγ protein expression in vitro [ 100 ]. In adipocytes, PPARγ alters the expression of fat metabolizing enzymes to increase FFA uptake into storage while simultaneously preventing the release of FFA [ 66 , 67 ]. However, in the β-cell some actions of PPARγ seem to mimic those of PPARα. Thus, one of the earliest demonstrations in islets of direct activation of PPARγ showed that TZD caused mobilization of triglyceride and increased FFA oxidation in Zucker diabetic fatty rats [ 101 ], resulting in improved insulin secretion [ 101 , 102 ]. This observation has been reinforced in more recent work. Induction of PPARγ by three different methods enhances expression of genes that participate in fatty acid oxidation [ 103 ]. Glucose-stimulated insulin secretion is enhanced by both PPARα and -γ agonists in db/db mice [ 104 ]. Consistent with this, mice with a partial global knockdown of PPARγ (PPARγ +/- ) on a high fat diet had blunted glucose-stimulated insulin secretion in isolated islets that was associated with an islet-specific accumulation of triglyceride [ 105 ] even though insulin resistance was partially prevented [ 106 ]. The TZD increase glucokinase and GLUT2 expression and activity via interaction with PPRE in the respective gene promoters [ 107 , 108 ]. A PPARα-agonist also induced GLUT2 expression in islets but the effect on glucokinase was not documented [ 109 ]. Improved glucose metabolism, however, has not been a consistent outcome of PPARγ induction [ 103 ]. Nonetheless, overexpression of PPARγ in a β-cell line is detrimental to glucose-stimulated insulin secretion and proinsulin synthesis, with PPARγ agonists causing a further negative effect [ 110 ]. Since PPARγ was not detected in control cells, it is unclear whether these results are physiologically relevant to primary β-cell function. Interestingly, in rodent islets PPARα is expressed at higher levels than PPARγ [ 76 ], while in human islets the situation is reversed [ 111 ]; the functions regulated by PPARα in rodents may be more pertinent to PPARγ in human β-cells. PPARγ activation also regulates some β-cell functions that have not been ascribed to PPARα. PPARγ activation by TZD may relieve oxidative stress in β-cells of diabetic animals [ 112 ], leading to preservation of β-cell mass [ 104 , 112 - 114 ] and partial improvement in glucose-stimulated insulin secretion from isolated islets [ 112 ]. The potential anti-oxidative or anti-inflammatory effects of TZD in islets of type 2 diabetes models are interesting in light of reports that TZD reduce diabetes incidence in non-obese diabetic (NOD) mice [ 115 ] and more generalized inflammatory/immune responses in a variety of tissues [ 116 ]. Moreover, PPARγ appears to be a critical determinant of β-cell expansion in response to a high fat diet [ 117 ]. However, despite these studies showing that PPARγ exists in β-cells and that its activation can regulate gene expression and cell function, Rosen et al. [ 117 ] recently showed that the TZD's antidiabetic effects are still fully present in mice in which PPARγ has been specifically eliminated only in β-cells. Thus, the dominant effects of dietary TZD on insulin secretion are likely indirect, a consequence of improved lipemia and glycemia. Sterol regulatory element binding protein The family of SREBPs governs transcriptional activation of a large number of genes involved in regulation of lipid metabolism, including lipogenesis, cholesterol transport and synthesis [ 118 ]. Of interest is the high expression of SREBP-1c in liver and adipose tissue [ 119 ], and its detection in pancreatic β-cells [ 120 ]. The primary function of SREBP-1c is to regulate transcription of genes involved in lipogenesis, such as acetyl-CoA carboxylase, fatty acid synthase and steroyl-CoA desaturase [ 119 ] and enzymes of glycolysis [ 118 , 119 ]. In the liver SREBP-1c appears to mediate the transcription of most insulin-responsive genes and in turn its expression, and possibly its activation, are induced by insulin [ 119 ]. Thus, SREBP-1c activity is enhanced during periods of dietary plenty; when glucose is abundant and insulin is stimulated. The outcome of SREBP-1c activation is to promote fat-sparing, leading to an increased synthesis of saturated and monounsaturated fatty acids, triglycerides and phospholipids, as well as enhanced glucose utilization via the glycolytic pathway [ 119 ]. Elevation of SREBP-1c in obesity characterized by hyperinsulinemia may therefore explain the onset of fatty liver. SREBP-1c appears to have a similar function in lipogenesis in pancreatic β-cells as in hepatocytes, but the effects on glycolytic enzymes have received little attention. Notably, blockade of SREBP-1c expression attenuates the glucose-induced increase in acetyl-CoA carboxylase activity seen in control β-cells [ 121 ] whereas an increase in SREBP-1c induces lipogenic enzymes, triglyceride accumulation and UCP2 expression [ 105 , 122 - 124 ]. The outcome of elevated SREBP-1c is a decrease in glucose metabolism and glucose-stimulated insulin secretion in all cases. Consistent with these studies utilizing molecular manipulation of SREBP-1c expression, studies of Zucker diabetic fatty rats demonstrate increased SREBP-1c levels in islets [ 120 ]. SREBP-1c has also been implicated as a regulator of apoptosis in β-cells [ 122 ]; thus the loss of β-cell mass seen in obese-diabetic models might be related to events triggered by this transcription factor. Indeed, β-cell apoptosis might be under control of both PPARγ and SREBP-1c because TZD has been reported to block the increase in SREBP-1c in diabetic fatty rats [ 120 ]; this implies that PPARγ regulates SREBP-1c. Conversely, other groups have evidence that SREBP-1c can up-regulate PPARγ mRNA expression [ 103 , 123 ] ; thus, the relationship between these two factors is not yet clear. The UCP2 promoter has a sterol response element [ 124 ] so the negative effects of SREBP-1c on insulin secretion might be caused by its induction of UCP2. However, reducing UCP2 expression by means of a small interfering RNA only partially restored glucose-stimulated insulin secretion in SREBP-1c-overexpressing cells. Likewise, activation of the AMP-activated kinase partially rescued the phenotype of the cells with SREBP-1c induction [ 125 ]. Certainly, SREBP-1c is implicated as a key contributor to lipotoxicity, as proposed elsewhere [ 89 , 126 ] but further research is required to fully understand its role in regulating insulin secretion in health and diabetes. Conclusions FFA exert dual effects on insulin secretion, dependent on the duration of exposure. Acute exposure to FFA increase glucose-stimulated insulin secretion whereas chronic exposure attenuate glucose sensitivity of pancreatic β-cells. The coordinated control of these processes by lipid-sensing transcription factors and its relevance to β-cell dysfunction in type 2 diabetes mellitus is increasingly a subject of investigation. PPARs (especially PPARα and PPARγ) are involved in the long-term regulation of lipid metabolism and their activity is modulated by endogenous lipid-derived ligands. PPAR agonists have positive effects on glucose homeostasis and lipid metabolism and can reduce cardiovascular events in obese-diabetic patients. PPARα is a fasting lipid oxidation-glucose sparing regulator whereas PPARγ is post-prandial lipid storing-glucose utilizing regulator. In islets, however, both PPARα and -γ appear to have some functions more consistent with PPARα, particularly induction of lipid oxidizing enzymes, which is potentially particularly important for maintaining basal insulin secretion. Growing evidence suggests that PPARγ is a regulator of β-cell proliferation and that PPARγ agonist-mediated anti-oxidative effects may also contribute to anti-diabetic activity. SREBP-1c up-regulates lipogenic enzymes in β-cells as it does in liver. Its chronic induction in islets of obese-diabetic rodents may therefore contribute to lipotoxicity by promoting triglyceride accumulation and removing fatty-acid derived signalling factors from the cellular pool. SREBP-1c and PPAR functions appear to be closely linked through cross-talk between the pathways that control their own expression, and may function in concert to affect not only fatty acid metabolism but also glucose metabolism, β-cell proliferation and apoptosis. Drugs given orally to activate PPARs can improve insulin sensitivity of peripheral tissues and generally appear to enhance β-cell function secondary to their insulin-sensitizing effects. However, it remains possible that specific effects on β-cells are also important contributors to the positive metabolic effects of PPAR agonists in type 2 diabetes treatment. Declaration of Competing Interests The author(s) declare that they have no competing interests. Authors Contributions ZF-H and CBC contributed equally to the writing of this review.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544854.xml
515366	A Drosophila Pattern Recognition Receptor Contains a Peptidoglycan Docking Groove and Unusual L,D-Carboxypeptidase Activity	The Drosophila peptidoglycan recognition protein SA (PGRP-SA) is critically involved in sensing bacterial infection and activating the Toll signaling pathway, which induces the expression of specific antimicrobial peptide genes. We have determined the crystal structure of PGRP-SA to 2.2-Å resolution and analyzed its peptidoglycan (PG) recognition and signaling activities. We found an extended surface groove in the structure of PGRP-SA, lined with residues that are highly diverse among different PGRPs. Mutational analysis identified it as a PG docking groove required for Toll signaling and showed that residue Ser158 is essential for both PG binding and Toll activation. Contrary to the general belief that PGRP-SA has lost enzyme function and serves primarily for PG sensing, we found that it possesses an intrinsic L,D -carboxypeptidase activity for diaminopimelic acid-type tetrapeptide PG fragments but not lysine-type PG fragments, and that Ser158 and His42 may participate in the hydrolytic activity. As L,D -configured peptide bonds exist only in prokaryotes, this work reveals a rare enzymatic activity in a eukaryotic protein known for sensing bacteria and provides a possible explanation of how PGRP-SA mediates Toll activation specifically in response to lysine-type PG.	Introduction Activation of innate immunity in response to bacterial pathogens requires a group of molecules, known as the pattern recognition receptors, that recognize conserved motifs, present in bacteria but absent in higher eukaryotes, and trigger downstream signaling events. In Drosophila, two distinct signal transduction pathways are involved in the pathogen-specific innate immune response by inducing the expression of a panel of specific antimicrobial peptides ( Tzou et al. 2002 ; Hoffmann 2003 ). The Toll signaling pathway responds mainly to Gram-positive bacterial or fungal infections, which lead to the proteolytic processing of the cytokine-like polypeptide Spätzle. Binding of the cleaved Spätzle to the transmembrane receptor Toll activates an intracellular signaling cascade that results in the degradation of the IκB-like protein Cactus and the nuclear localization of the NF-κB–like proteins Dif and Dorsal, which induce the transcription of several antimicrobial peptide genes, such as Drosomycin ( Lemaitre et al. 1996 , 1997 ; Meng et al. 1999 ; Rutschmann et al. 2000b ; Tauszig-Delamasure et al. 2002 ; Weber et al. 2003 ). By contrast, the immune deficiency (Imd) pathway mediates defense reactions against primarily Gram-negative bacteria through different signaling components and regulates the cleavage and activation of another NF-κB–related nuclear factor, Relish, which activates a different set of antimicrobial peptide genes, including Diptericin ( Lemaitre et al. 1995 ; Hedengren et al. 1999 ; Leulier et al. 2000 ; Rutschmann et al. 2000a ; Vidal et al. 2001 ). Several genetics studies have shown that the Toll pathway and the Imd pathway are activated specifically by two distinct peptidoglycan recognition proteins (PGRPs) in response to bacterial infections ( Michel et al. 2001 ; Choe et al. 2002 ; Gottar et al. 2002 ; Ramet et al. 2002 ). PGRPs constitute a highly diversified family of proteins present in both insects and mammals. Members of the PGRP family are expressed as either secreted, cytosolic, or transmembrane forms, which all share a conserved 165-amino acid domain (the PGRP domain) with an evolutionary connection to bacteriophage T7 lysozyme ( Yoshida et al. 1996 ; Kang et al. 1998 ; Ochiai and Ashida 1999 ; Werner et al. 2000 ; Liu et al. 2001 ). There are 13 PGRP genes in the genome of Drosophila ( Werner et al. 2000 ). Remarkably, a gene knockout of PGRP-SA, an extracellular protein, is sufficient to eliminate Toll activation in response to the Gram-positive bacterium Micrococcus luteus in adult flies ( Michel et al. 2001 ). Similar loss-of-function screenings have identified PGRP-LC as the surface transmembrane receptor for the Imd pathway, although another PGRP member, PGRP-LE, may also be involved in Imd activation ( Choe et al. 2002 ; Gottar et al. 2002 ; Ramet et al. 2002 ; Takehana et al. 2002 ; Werner et al. 2003 ). Several PGRPs have been shown to bind peptidoglycan (PG) ( Yoshida et al. 1996 ; Werner et al. 2000 ; Takehana et al. 2002 ; Kim et al. 2003 ), an essential and unique cell-wall polymer found in both Gram-positive and Gram-negative bacteria. PG is composed of long glycan chains made of two alternating sugars and cross-linked by short peptides. The subunits of PG, also known as muropeptides, are composed of N -acetyl glucosamine (GlcNAc) and N -acetyl muramic acid (MurNAc) plus a stem peptide chain consisting of D - and L - (or meso -) amino acids, with the third amino acid being most frequently lysine in Gram-positive bacteria and diaminopimelic acid (DAP) in Gram-negative bacteria. Recently, Leulier and colleagues (2003) have shown that the Toll pathway is activated primarily by lysine-type PG found in most Gram-positive bacteria but responds weakly to DAP-type PG from Gram-negative bacteria. Not only did this finding reinforce the identification of PGRP-SA and PGRP-LCs as the putative receptors of the Toll and Imd pathways for bacterial molecular patterns, respectively, it also suggested that the signaling specificities of these two pathways might rely on the binding capability of the two activating PGRPs towards specific PG forms. Results/Discussion To facilitate molecular characterization of PG recognition and signal transduction mediated by PGRP-SA, we overexpressed and purified recombinant PGRP-SA (rPGRP-SA) in a baculovirus-insect cell expression system. PGRP-SA is a secreted protein circulating in the hemolymph (the insect blood) of Drosophila. We tested the activity of rPGRP-SA in vivo by injecting the protein into wild-type (wt) and PGRP-SA–deficient (PGRP-SA seml ) flies. For this assay we used flies carrying a Drosomycin-GFP reporter transgene, which served as the target gene of the Toll signaling pathway. The wt flies injected with water produced Drosomycin-GFP after challenge by M. luteus, whereas PGRP-SA seml flies failed to express the reporter gene after the same treatment ( Figure 1 A and 1 B). When 112 ng of rPGRP-SA was injected into PGRP-SA seml flies, the recipient flies became capable of producing Drosomycin-GFP after challenge with M. luteus ( Figure 1 C). As little as 11 ng of rPGRP-SA was sufficient to rescue PGRP-SA seml flies ( Figure 1 D). Injection of 11 ng of rPGRP-SA in wt and PGRP-SA seml flies without any further microbial challenge could not activate Drosomycin-GFP expression (unpublished data). These results demonstrate that rPGRP-SA expressed in insect cell culture medium is active in vivo. Figure 1 The In Vivo Rescuing and In Vitro PG-Binding Activities of Wild-Type rPGRP-SA (A–E) Drosomycin-GFP expression in (A) wild-type and (B to E) PGRP-SA seml flies after challenge by M. luteus. (A and B) Water or (C to E) rPGRP-SA at variable concentrations was injected into Drosomycin-GFP flies prior to the challenge with M. luteus. (F) rPGRP-SA binds to both lysine-type (M. luteus and E. faecalis) and DAP-type (E. coli and P. aeruginosa) PGs but not to amidated DAP-type (Bacillus subtilis and Bacillus thuringiensis) PG. The left lane (Input) is loaded with the same amount (20 μg) of protein used for the binding assay. The selective activation of the Toll and Imd pathways by distinct classes of bacteria is mediated via recognition of specific forms of PGs ( Leulier et al. 2003 ). We analyzed the PG binding of rPGRP-SA to test whether the differential activation of Toll by different PG forms reflects their different binding ability towards PGRP-SA. We found that rPGRP-SA binds to purified lysine-type PGs from M. luteus or Enterococcus faecalis and to DAP-type PGs from Escherichia coli or Pseudomonas aeruginosa, but not to amidated DAP-type PGs from Bacillus ( Figure 1 F). Although the sensitivity of this assay is insufficient to compare the differential binding of rPGRP-SA to lysine- and DAP-type PGs, these results are overall in good agreement with previous in vivo challenge data ( Leulier et al. 2003 ). Unlike lysine-type PGs, DAP-type PGs can only weakly activate the Toll pathway, which may be explained by the unexpected hydrolyzing activity of PGRP-SA for DAP-type PGs, as described below. We crystallized PGRP-SA using seeding methods, and collected complete data to 2.2-Å resolution from a single crystal plate at the SBC 19-ID beamline of the Advanced Photon Source (APS) at Argonne National Laboratory. The crystal structure was determined by molecular replacement, using the structure of PGRP-LB as a search model. PGRP-LB is a zinc amidase similar in structure to T7 lysozyme ( Kim et al. 2003 ) and is 29% identical in amino acid sequence to PGRP-SA. Although the crystals were grown from full-length PGRP-SA (177 residues plus 6×His-tag; residues numbered from the N terminus of the purified polypeptide chain, as determined by N-terminal sequencing), clear electron density was visible only from Cys11 to Pro177. The structure of PGRP-SA reveals a single domain composed of a central seven-stranded mixed β sheet (B1, B3, B4, B5, B7, B8, and B9) flanked by three major helices (H2, H3, and H5), a small two-stranded parallel β sheet (B2 and B6), and two single-turn helices (H1 and H4) ( Figure 2 A). The H2 helix contains one turn of rarely observed π helix at its C terminus (residues 64–70). This helix, together with the L1–L4 loops and the central β sheet, forms a prominent extended surface groove ( Figure 2 A and 3 ), which in PGRP-LB includes a zinc cage ( Kim et al. 2003 ). The overall structure of PGRP-SA strongly resembles that of PGRP-LB ( Figure 2 B). The root-mean-square deviation (r.m.s.d.) of the 167 Cα positions after superposition is 1.22 Å. However, PGRP-SA has lost two of the four zinc-coordinating residues present in PGRP-LB ( Figure 2 C); accordingly, the rPGRP-SA crystals exhibit no X-ray absorption at the zinc edge, and we found no electron density for possible metal ions around the groove. Other major differences are located in the N and C termini, the loop immediately preceding B3, the B4-B5 β-hairpin, and the L1 loop ( Figure 2 B), where the sequences among PGRPs are highly diverse ( Figure 2 C). PGRP-SA contains two disulfide bridges (Cys11-Cys134 and Cys48-Cys54), whereas PGRP-LB has only one. The highly conserved disulfide bridge Cys48-Cys54 is the target of the PGRP-SA seml mutation in which Cys54 is changed into a tyrosine ( Michel et al. 2001 ). The Cys48-Cys54 bridge tethers the H2 helix to the central β sheet through the L1 loop ( Figure 2 A). The other disulfide bond, between Cys11 and Cys134, is solvent exposed and anchors the N-terminal portion of PGRP-SA onto the H3 helix. As the C terminus of the protein is also tethered by insertion of the proline ring of the terminal residue Pro177 into a hydrophobic pocket formed by Ile148 of B8 and Val153 of H4, the structure of PGRP-SA appears to be more compact than that of PGRP-LB. The integral domain structure of PGRP-SA may be required for protein stability, considering that PGRP-SA is an extracellular protein secreted into the Drosophila hemolymph. The disulfide bridge Cys11-Cys134 may also be present in several mouse and human PGRPs ( Figure 2 C). Figure 2 PGRP-SA Structure and Sequence Comparisons (A) Ribbon diagram showing the front view (left) and side view (right) of PGRP-SA. The ribbon is colored from N to C terminus in a progression from blue to red. Disulfide bridges are shown as sticks. The π helix turn at the end of the H2 helix is indicated. (B) Comparison of PGRP-SA (blue coil, from N to C) and PGRP-LB (green coil, from N′ to C′). (A) and (B) were prepared with Bobscript ( Esnouf 1999 ), GL_RENDER (E. Esser, personal communication), and POV-Ray (Persistence of Vision Ray Tracer v3.1g). (C) Aligned sequences of selected PGRP domains, with a serine and a histidine at position 158 and position 42 of PGRP-SA (marked with asterisks), respectively, from Drosophila (d), mouse (m), and human (h). Secondary-structure elements in PGRP-SA are indicated above the alignment. Invariant residues are boxed in black and colored in white, conserved residues are shaded in yellow, and those lining the putative PG docking groove are in pink. The disulfide bond-forming Cys residues are boxed in gray. The residue number of PGRP-SA is shown above the alignment. The residues chosen for mutagenesis are marked with black circles. A structurally based alignment of the dPGRP-LB sequence is shown at the bottom with its amidase catalytic zinc-coordinating residues colored in red. Figure 3 Structural Analysis of PGRP-SA (A and B) Molecular surfaces of (A) PGRP-SA and (B) PGRP-LB shown in similar orientations. Selected PGRP-SA residues on the putative PG docking groove are highlighted on the surface. Thr158 of PGRP-LB, the residue corresponding to Thr156 of PGRP-SA, is highlighted for reference. (C) Stick model of the PGRP-SA residues chosen for mutational analysis. Residues are colored with the same rainbow-coloring scheme as in Figure 2 A. Figures were prepared with GRASP ( Nicholls et al. 1991 ), Bobscript, GL_RENDER, and POV-Ray. The most prominent feature of PGRP-SA is a long surface groove demarcated by residues of the H2 helix from one side and of the L1–L4 loops from the other side, with the residues from B3, B4, and B7 of the central β sheet forming the base of the groove ( Figure 2 A). These residues are among the least conserved, and even the lengths of the L1 and L3 loops vary among members of the PGRP family ( Figure 2 C). Therefore, the surface groove structure of PGRP-SA is distinct from that of PGRP-LB despite their overall structural similarity ( Figure 3 A and 3 B). The presence of a surface groove on PGRP-SA suggests that it may have a role in PG binding. We performed mutagenesis and functional analysis to test this hypothesis. In the following text, mutations are identified by the one-letter code for the residue in wild-type PGRP-SA, followed by the position of the residue in the amino acid sequence and the one-letter code for the residue to which it was mutated (e.g., S158A has serine in position 158 mutated to alanine). Residues on the surface groove whose side chains are solvent accessible were chosen for mutational analysis (see Figure 2 C, 3 A, and 3 C). These residues are located in three different subregions of the putative docking groove. The first group of residues constitutes the right-side wall of the groove, based on the front view shown in Figure 3 (Tyr64, His65, Asp70, Phe71, and Asn72). The second group is located on the left-side wall of the groove (Val44, Thr45, Tyr100, Ile154, and Ser158). The last group includes Ser75, which sits at the base of the groove. Based on the structure, we also made a Thr-to-Tyr mutation for residue 156; we reasoned that the introduced bulky side chain of Tyr would prevent access of PG to the putative docking groove. In addition, the single mutation I14A, located on the backside of the molecule, was made as a control. None of the residues chosen for mutagenesis is involved in extensive packing interactions. Hence, alterations of these residues are not expected to disrupt the tertiary structure of PGRP-SA. Our hypothesis was that, if the surface groove is indeed involved in PG recognition, the Ala mutations within the groove should exhibit reduced or altered PG-binding activities, whereas the T156Y mutation should completely abolish PG interaction. We analyzed the ability of these single- or multiple-Ala mutants to bind lysine-type PG from M. luteus by in vitro PG-binding assays ( Figure 4 A). The in vivo activity of these rPGRP-SA mutants was examined by analyzing their capacity to rescue the PGRP-SA seml mutation in the assay described earlier; in addition, the Drosomycin expression was measured by quantitative real-time PCR analysis ( Figure 4 B). These studies showed that mutations at almost every position tested on both walls of the groove region led to impaired PG binding and Toll signaling activity except the S75A mutant, which exhibited an enhanced PG-binding ability. The T156Y mutation, as expected, resulted in a complete loss of the interaction with PG ( Figure 4 A); as a result, the mutant protein failed to activate the Toll pathway ( Figure 4 B). Notably, the single mutations S158A and S158C also completely abolished the function of the protein both as a PG recognition receptor and as a Toll activator. The enhanced activity on both PG binding and Toll activation of the S75A mutants suggests that the removal of the hydroxyl group of Ser75 may create a better binding surface for the PG. In fact, Ala and Gly are commonly found at this position in the sequences of PGRPs (see Figure 2 C). As expected, the I14A mutation on the backside of the molecule did not affect PG binding or Toll activation (unpublished data). It is interesting that some mutants of PGRP-SA with apparent PG-binding deficiency, for example Y100A and V44A/T45A, could still induce Drosomycin expression upon injection in response to challenge with PG from M. luteus. This discrepancy may be the result of different sensitivities between the gel-based PG-binding assay, which examines plain physical interaction between PGRP-SA and PG, and the rescue assay, by which the amplified signaling outcome of PG interaction with PGRP-SA, namely Drosomycin expression, is observed. Nevertheless, these results together indicate that the surface groove of PGRP-SA mediates both interaction with PG and activation of the Toll pathway. These studies further underscore the role of PGRP-SA as a true pattern recognition receptor, as they demonstrate the correlation between the biochemical recognition of PG and Toll activation through PGRP-SA. Figure 4 Mutational Analysis of PGRP-SA (A) Upper panel shows the wild-type and mutant rPGRP-SA pulled down by lysine-type PG from M. luteus. Lower panel (Input) shows the corresponding protein samples (20 μg) without incubation with PG. (B) The relative Toll signaling activities of the rPGRP-SA mutants. At least three repeats were performed for each experiment. Each bar represents the mean with the standard deviation. The values obtained for the wild type after M. luteus PG injection were arbitrarily set to 100 (upper dashed line). The background activity level is indicated by the lower dashed line. CTR, unchallenged control. The PGRP-SA seml mutation results in a Cys-to-Tyr mutation at position 54, which is engaged in a highly conserved Cys48-Cys54 disulfide linkage ( Michel et al. 2001 ). We conducted mutational analysis to investigate whether the PGRP-SA seml mutation eliminates PGRP-SA function by disrupting the conserved disulfide bridge and thus affecting the protein stability, or by sterically blocking the surface groove with the bulky side chain of Tyr. We found that the C48A mutant failed to be expressed in the insect cell culture, in which all the other wild-type and mutant proteins were expressed (unpublished data). As a result, the culture medium from the C48A mutant failed to restore the PGRP-SA seml phenotype after injection in Drosophila ( Figure 4 B). By contrast, the C11A mutation, which disrupts the Cys11-Cys134 disulfide bond on the backside of the molecule, had little effect on the function of the protein ( Figure 4 B). These results suggest that the PGRP-SA seml mutation disrupts the proper folding of PGRP-SA rather than PG interaction via the docking groove. It is intriguing that both of the single mutants, S158A and S158C, fail to bind lysine-type PG and to activate Toll. The S158 residue is located on one wall of the docking groove. Seven of the Drosophila PGRPs, including PGRP-SC1B and PGRP-LB, have been suggested to possess amidase activity. These “amidase PGRPs” all have a Cys residue at this position, which appears to participate in the Zn coordination in the active site. Substitution of Cys to Ser or Ala in PGRP-SC1B eliminates its enzymatic activity but not its capacity to bind PG, suggesting that the Cys is required for amidase activity ( Mellroth et al. 2003 ). Our data show that, in PGRP-SA, Ser158 is essential for its interaction with PG. Also, Ser158 is highly conserved among PGRPs that have lost the amidase catalytic residues (see Figure 2 C). The drastic loss-of-function effect of S158A and S158C mutations suggests that the chemical property of the Ser side chain at this position may be critical for function. Recently, muropeptides have been identified as the bacterial molecular patterns detected by Nod proteins ( Chamaillard et al. 2003 ; Girardin et al. 2003a , 2003b ; Inohara et al. 2003 ). Free muropeptides are found within bacterial cells as they are constantly synthesized de novo or hydrolyzed from PG and recycled during cell division ( Goodell 1985 ; Goodell and Schwarz 1985 ); they could be released from bacterial cells during infection and exploited for bacterial sensing by pattern recognition molecules in the host. PGRPs are structural homologues of the N -acetylmuramoyl- L -alanine amidase superfamily of proteins, including AmpD and T7 lysozyme, which can hydrolyze monomeric muropeptides or larger PG fragments ( Inouye et al. 1973 ; Kang et al. 1998 ; Liepinsh et al. 2003 ; Mellroth et al. 2003 ). Therefore, muropeptides or their peptidic moieties may serve similarly as specific ligands for PGRP-SA and PGRP-LC via interaction with the PG docking groove. Accordingly, our structural modeling has indicated that the structure of the long docking groove on PGRP-SA is able to fit a ligand with elongated conformation, which a muropeptide or its stem peptide could adopt (unpublished data). Previously, PGRP-SC1B and PGRP-LB have been demonstrated to display a T7 lysozyme-like amidase activity ( Kim et al. 2003 ; Mellroth et al. 2003 ). We sought to determine if rPGRP-SA has any enzymatic activity, although it has been believed not to possess such an activity; PGRP-SA is missing a critical cysteine residue found in the active site of these amidase PGRPs ( Mellroth et al. 2003 ) (see Figure 2 C). We incubated rPGRP-SA separately with either the lysine-type muropeptide, GlcNAc-MurNAc(anhydro)- L -Ala-γ- D -Glu- L -Lys- D -Ala, or the corresponding DAP-type muropeptide, GlcNAc-MurNAc(anhydro)- L -Ala-γ- D -Glu- meso -DAP- D -Ala, and analyzed the reaction mixtures afterwards by high performance liquid chromatography (HPLC) ( Figure 5 ). To our surprise, we observed within 40 h of incubation a near-complete cleavage of the DAP-type muropeptide, but not the lysine-type compound, at a specific peptide bond position, resulting in a product consisting of the tripeptide derivative GlcNAc-MurNAc(anhydro)- L -Ala-γ- D -Glu- meso -DAP, with the release of the terminal D -Ala ( Figure 5 B to 5 E). These results demonstrated that rPGRP-SA had cleaved between the meso -DAP at position 3 and the D -Ala at position 4 on the stem peptide and thus exhibited an L , D -carboxypeptidase activity. Typical Michaelis-Menten kinetics were observed in the substrate concentration range considered (10–500 μM). The K m value of rPGRP-SA for its substrate GlcNAc-MurNAc(anhydro)- L -Ala-γ- D -Glu- meso -DAP- D -Ala was 21.4 ± 1.8 μM and the catalytic constant k cat was 0.48 ± 0.02 h −1 . The small turnover number of PGRP-SA estimated for hydrolyzing DAP-PG substrates is comparable to the k cat of small G proteins such as Ras GTPases. We also tested different other PG-related tetrapeptide compounds as substrates and found that rPGRP-SA hydrolyzed the same peptide bond in DAP-containing muropeptides but had no detectable activity on all the lysine-type compounds tested (unpublished data). This enzymatic activity of rPGRP-SA was not inhibited by ethylenediaminetetraacetic acid (EDTA) or by phenylmethylsulfonyl fluoride (PMSF) (unpublished data). We observed that the two Ser158 mutants, S158C and S158A, did not exhibit any detectable activity ( Figure 5 F and unpublished data), although both bind DAP-PG from E. coli as wild-type rPGRP-SA does ( Figure 6 A). In the PGRP-SA structure the Oγ of the Ser158 residue is positioned within hydrogen-bonding distance (2.95 Å) of the Nδ1 of the highly conserved His42 residue ( Figure 6 B). The fact that the enzymatic activity of rPGRP-SA can be completely eliminated by removing the hydroxyl group of Ser158 (S158A) or by replacing it with a thiol group (S158C) suggests that Ser158 is involved in catalysis rather than in the binding of the DAP-containing substrates. In fact, a Ser-His catalytic dyad of a catalytic antibody was found to be sufficient for catalyzing the hydrolysis of amino acid esters ( Zhou et al. 1994 ). To test this hypothesis, we generated a H42A mutant and analyzed its enzymatic activity. Indeed, this mutant is incapable of hydrolyzing DAP-peptide substrate, although it preserves the ability to bind DAP-PG (see Figure 5 G and 6 A). Therefore, this result supports the catalytic role of the S158-H42 dyad for the hydrolyzing activity of PGRP-SA. Our enzymatic analysis data show that rPGRP-SA is a carboxypeptidase with an apparent specificity for the L,D -configured DAP-peptide bond between the carboxyl group at the L -center of the meso -DAP and the amino group of the following D -Ala residue. Through showing that DAP-containing muropeptides are the substrates for the PGRP-SA enzyme, we provide biochemical evidence suggesting that PGRP-SA may recognize a specific monomeric PG fragment. In support of this finding, it has been demonstrated very recently that specific monomeric DAP-PG fragments can activate the Imd pathway via PGRP-LC in flies and in cell culture ( Kaneko et al. 2004 ). Figure 5 PGRP-SA Is an L,D -Carboxypeptidase (A) Chemical structures of the DAP-type muropeptide, GlcNAc-MurNAc(anhydro)- L -Ala-γ- D -Glu- meso - DAP - D -Ala, and lysine-type muropeptide, GlcNAc-MurNAc(anhydro)- L -Ala-γ- D -Glu- L - Lys - D -Ala, used in the enzymatic assays (substrates S 1 and S 2 , respectively). The arrow indicates the site of cleavage of the DAP-type substrate S 1 by the L,D -carboxypeptidase activity. (B–G) Reverse-phase HPLC analysis. Cleavage of the DAP-type substrate S 1 by wild-type rPGRP-SA results in the generation of GlcNAc-MurNAc(anhydro)- L -Ala-γ- D -Glu- meso -DAP (P 1 product). The position of the peak corresponding to standard GlcNAc-MurNAc(anhydro)- L -Ala-γ- D -Glu- L -Lys (P 2 product, not generated by rPGRP-SA) is indicated. (B) Incubation of S 1 without rPGRP-SA for 40 h. (C and D) Incubation of S 1 with rPGRP-SA for (C) 24 h and (D) 40 h. (E) Incubation of S 2 with rPGRP-SA for 70 h. (F and G) Incubation of S 1 with the (F) S158C and (G) H42A mutants for 40 h. Figure 6 DAP-Type PG-Binding Activities of the S158A/C and H42A Mutants and the Structure of the S158-H42 Dyad in the Active Site (A) Upper panel shows the wild-type and mutant rPGRP-SA pulled down by DAP-type PG from E. coli. Lower panel (Input) shows the corresponding protein samples (20 μg) without incubation with PG. (B) Stereo diagram showing the putative active-site residues. Prepared with Bobscript, GL_RENDER, and POV-Ray. So far, only one L,D -carboxypeptidase, from E. coli, has been identified and characterized ( Ursinus and Holtje 1994 ; Templin et al. 1999 ). Hence, we report here the first eukaryotic protein exhibiting such an activity specific for peptide bonds existing only in prokaryotes. The DAP-PG hydrolyzing activity of PGRP-SA has a rather slow turnover number. It would be interesting to see if this low intrinsic DAP-PG hydrolyzing activity could be stimulated by another hemolymph protein(s) that could associate with PGRP-SA, such as Gram-negative bacteria-binding protein 1 (GNBP1) (see below). Our observation that a pattern recognition receptor has enzymatic activity is unexpected. However, as rPGRP-SA binds to both lysine-type and DAP-type PGs (see Figure 1 F), this finding suggests that the specific activation of Toll by lysine-type PG is achieved by the concomitant ability of PGRP-SA to recognize lysine-type PG and to hydrolyze DAP-type PG. Since the latter has been identified as a strong inducer of the Imd pathway, it will be interesting to see whether the L,D -carboxypeptidase activity of PGRP-SA can influence the Imd pathway positively by generating specific PG fragments that are recognized by PGRP-LC or negatively by scavenging DAP-PG to eliminate its immune-elicitor activity. As PGRP-LCx/a, the other known pathogen-sensing receptors, and several other PGRPs also possess a Ser residue at the position equivalent to S158 in PGRP-SA (see Figure 2 C), it will be interesting to see if they possess similar enzymatic activity. In the present study, we characterized the PG docking groove of PGRP-SA through a combined structural and functional analysis, and we showed that this surface groove mediates both PG sensing and Toll signaling and that Ser158 in the groove is involved in PG interaction, Toll activation, and the newly discovered L,D -carboxypeptidase activity. The S158C mutation has a dramatic negative effect on the ability to activate the Toll pathway. This suggests that the hydroxyl group of Ser158 may mediate critical interaction, perhaps via a hydrogen bond, with a specific lysine-type PG fragment and that this interaction may contribute the bulk of the binding energy. However, we found that the same mutation did not affect binding to DAP-type PG, suggesting that Ser158 is not critical for DAP-type PG interaction ( Figure 6 A). Therefore, PGRP-SA appears to employ different binding modes for interactions with lysine-type PG versus DAP-type PG. meso -DAP differs from L -lysine only by the substitution of a carboxyl group on the Cɛ with D -chirality. As the carboxypeptidase activity of PGRP-SA can act only on DAP-type and not on lysine-type PG compounds, it is likely that the carboxyl group at the D -center of DAP provides the critical interaction(s) with the docking groove residue(s) to help orient the peptide bond between DAP and D -Ala. However, understanding the structural basis of the selectivity to DAP-PG over lysine-PG would require a cocrystal structure of PGRP-SA and a lysine-type PG ligand. As the PG docking groove is lined with residues that are highly diverse among different PGRPs, indicating that each PGRP protein may bind to a specific set of PG fragments, determining the structure of PG ligand-bound PGRP-SA will also provide important insights into PG recognition specificity of this family of proteins. However, so far, no cocrystal of a PGRP protein with a PG compound has been obtained. Drosophila possesses a high number of genes encoding serine proteases and serine protease inhibitors (serpins) ( Rubin et al. 2000 ). Serine protease cascades, operating through sequential zymogen activation, have been implicated in dorsal-ventral fate determination and hemolymph clotting in arthropods ( Krem and Cera 2002 ). A hemolymph serine protease (Persephone) has been shown to mediate the cleavage of Spätzle in response to fungal infection ( Ligoxygakis et al. 2002 ). As PGRP-SA is not involved in fungal-dependent cleavage of Spätzle and activation of Toll, it is believed that this hemolymph pattern recognition protein activates another unidentified proteolytic enzyme(s), resulting in the cleavage of Spätzle specifically in response to bacterial infection. Recently, another hemolymph protein, GNBP1, has been shown to critically participate in activating Toll, perhaps by associating with PGRP-SA, in response to Gram-positive bacterial infection ( Gobert et al. 2003 ; Pili-Floury et al. 2004 ). Based on our result indicating that PGRP-SA may recognize monomeric PG ligands, it is likely that docking of the specific PG compound onto the surface groove of PGRP-SA may create a new molecular surface that would allow interaction with other Toll-activating factors such as GNBP1. Furthermore, perhaps a multiprotein complex involving PG ligand-bound PGRP-SA and GNBP1 is involved in direct proteolytic activation of the upstream protease of a Spätzle-processing protease cascade. Alternatively, a PG-dependent PGRP-SA/GNBP1 complex may be involved in binding and sequestering a serpin to release the inhibition of the Spätzle-processing enzyme cascade. A hemolymph serpin (Necrotic) has been implicated in inhibiting the proteolytic cleavage of Spätzle upon fungal infection ( Levashina et al. 1999 ). Although a better understanding of PGRP-SA/GNBP1-activated cleavage of Spätzle will require identification of critical players that link the microbial recognition to the proteolytic activation of Spätzle, more detailed biochemical and structural studies on the minimal PG moiety recognized by PGRP-SA and the interaction between PGRP-SA, its specific PG ligand, and GNBP1 are necessary to help define the molecular mechanism of PG recognition mediated by these pattern recognition receptors. Materials and Methods Protein expression, purification, and crystallization Details on the cloning, expression, purification, and crystallization of recombinant Drosophila PGRP-SA will be presented elsewhere. Briefly, full-length PGRP-SA (including its N-terminal signal peptide) with a 6×His tag at the C terminus was overexpressed in insect Hi-5 cells using the Bac-to-Bac baculovirus expression system (Invitrogen, Carlsbad, California, United States) and purified with Talon metal affinity resins (Clontech, Palo Alto, California, United States) followed by size exclusion on a Superdex 75 column (Pharmacia, New York, New York, United States) pre-equilibrated in 20 mM Tris-HCl (pH 7.8) and 300 mM NaCl. The purified protein was analyzed by N-terminal sequencing and mass spectrometry to ensure its identity and purity. Crystallization was carried out at 21 °C by the hanging-drop vapor diffusion technique. The protein formed plate-like clusters over a reservoir containing 2.0 M NaKPO 4 (pH 6.2). Single crystals were produced by two successive rounds of streak- and macroseeding and were cryoprotected in reservoir solution supplemented with 30% glycerol before data collection. Structure determination We collected X-ray diffraction data using synchrotron radiation at the 19-ID beamline at APS. The diffraction images were processed and scaled with the HKL2000 package ( Otwinowski and Monor 1997 ). The positions of the two molecules in the asymmetric unit were determined by molecular replacement with the program AmoRe ( Navaza 1994 ) using the PGRP-LB structure as the search model (PDB code 1OHT). The two solutions were related by rotation and translation operations, generating a nonsymmetric dimer. The current model was refined after iterative cycles of manual rebuilding with the program O ( Jones et al. 1991 ) and refinement with the program CNS ( Brunger et al. 1998 ) ( Table 1 ). The PGRP-SA dimer in the crystal is probably not biologically relevant, as it was not revealed by gel-filtration chromatography; moreover, the dimer interface was found to involve several phosphate ions from the crystallizing reagent and the first His residue of the affinity tag from one of the monomers. Table 1 Data Collection and Refinement Statistics a R merge = Σ\|( I hkl ) − < I >Σ\|/( I hkl ), where I hkl is the integrated intensity of a given reflection b R = (Σ\| F o − F c \|)/(Σ F o ), where F o and F c are observed and calculated structure factors, respectively c Calculated using the program PROCHECK ( Laskowski et al. 1996 ) Site-directed mutagenesis Point mutations were generated by a PCR-based strategy using the QuikChange Kit (Stratagene, La Jolla, California, United States), and the identities of the mutagenized products were verified by sequencing. Fly stocks and protein microinjection y, w, P( ry + , Diptericin-lacZ ), P( w + , Drosomycin-GFP ) flies were used as wild-type strains ( Manfruelli et al. 1999 ). Drosomycin-GFP, PGRP-SA seml is a line carrying the semmelweis mutation in PGRP-SA (C54Y) ( Michel et al. 2001 ). Drosophila stocks were maintained at 25 °C with standard medium. A quantity of 9.2 nl of water or rPGRP-SA protein was injected into the thorax of wild-type or PGRP-SA seml female adults (3–4 d old) using a Nanoject apparatus (Drummond, Broomall, Pennsylvania, United States). One hour later, flies were infected with a thin needle previously dipped into a concentrated culture of M. luteus or given an injection of 9.2 nl of M. luteus PG (5 mg/ml). Flies were then incubated for 24 h at 25 °C. A highly purified solution of M. luteus PG was produced and injected in flies as described by Leulier and colleagues (2003) . Quantitative real-time PCR For Drosomycin quantification from whole animals, RNA was extracted using RNA TRIzol (Invitrogen). cDNAs were synthesized using SuperScript II (Invitrogen) and PCR was performed using dsDNA dye SYBR Green I (Roche Diagnostics, Basel, Switzerland) on a Lightcycler (Roche). All samples were analyzed in duplicate and the amount of mRNA detected was normalized to control Rp49 mRNA values. We used normalized data to quantify the relative levels of a given mRNA according to cycling threshold analysis (ΔCt). PG-binding assay The assay was performed at 4 °C by incubating 20 μg of purified wild-type or mutant rPGRP-SA with 300 μg of insoluble PGs, prepared as described previously ( Leulier et al. 2003 ), in 300 μl of binding buffer containing 20 mM Tris-HCl (pH 7.8) and 300 mM NaCl on a shaking platform for 1 h. Bound protein, retained in the PG pellet after spinning the incubation mixture at 16,000 × g for 5 min, was washed with 1 ml of binding buffer followed by a 5-min spin and finally dissolved in 10 μl of SDS buffer for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The PG-bound rPGRP-SA was visualized by Coomassie Blue staining. Enzymatic assay and reverse-phase HPLC analysis The activity was tested in 20 mM HEPES (pH 7.4) containing 2.5 mM EDTA, 50 μM substrate, and enzyme (25 μg of wild-type or mutant rPGRP-SA) in a total volume of 50 μl. After incubation for the indicated period at 37 °C, the mixture was injected on a Nucleosil 100 C 18 5μ reverse-phase HPLC column (4.6 mm × 250 mm, Alltech France, Templemars, France) and elution was performed at 0.6 ml/min with buffer A (50 mM sodium phosphate [pH 4.45]) for 10 min and then with a gradient of methanol in buffer A (from 0% to 25% in 50 min). Peaks were detected at 215 nm. In all cases, substrates and products were purified and desalted by HPLC, and their identity was confirmed by amino acid and mass spectrometry analyses. Supporting Information The atomic coordinates and structure factors have been deposited in the Protein Data Bank ( http://www.rcsb.org/pdb/ ) under accession number 1S2J.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC515366.xml
406520	Turning Down the Volume: Why Some Genes Tolerate Less Noise	null	All organisms have evolved complex mechanisms designed to exquisitely regulate the expression of appropriate genes at their correct levels. Natural random variation in the processes of regulation and expression, however, limits the precision with which protein production can be controlled. This subtle variation, or “noise,” in the expression of genes has been studied with increasing interest. Though much progress has been made in understanding the amount of noise that exists and the cellular processes that underlie it, the physiological impact of noise, and whether it is biologically relevant or can just be ignored, has been less clear. “Shhhhhhhhhhhhhh!” (Photo by Bryan Zeitler and Jennifer Zeitler) To answer this question, Hunter Fraser et al. asked whether noise in gene expression exerts an equal effect on all genes in the genome. Is noise in gene expression irrelevant to the fitness and well-being of cells, or do cells need to minimize noise in the expression of some or all genes? If noise in gene expression has a negative impact on cells, they reasoned, that impact should vary from gene to gene depending on the gene's function. There should be selection to minimize noise for those genes most crucial to cell survival and function. Thus, a genome-wide analysis of noise in gene expression, they predicted, would show that genes for which “noisy” expression would be most harmful would display less of it. The researchers examined this question in the budding yeast Saccharomyces cerevisiae because of the vast quantity and variety of genomic data available for this organism. Previous research has shown that the noise that exists in the expression of a gene is directly related to the rates of transcription and translation. Using data available from previous genome-wide studies, the authors were able to estimate these rates, and therefore the noise, for nearly every gene in the yeast genome. After estimating the amount of noise in the expression level for nearly every gene, the authors examined two subsets of genes that they hypothesized would be particularly affected by noise. First they looked at “essential” genes, reasoning that since total lack of expression of these genes results in death, even small variations in expression resulting from noise would often exert a negative impact. Previous research had identified all the essential genes in yeast by deleting each gene individually and assessing the fitness of the resulting mutant. Here, the authors compared the levels of noise in this pool of essential genes to that of nonessential genes. They found that essential genes usually display less noise than nonessential genes, lending support to their hypothesis. They similarly examined genes encoding proteins involved in forming multiprotein complexes. Because these complexes are built of proteins in specific ratios, over- or under expression of one component will hinder the accurate assembly of productive complexes. So a high degree of noise could interfere with the coordinated expression necessary for proteins involved in these complexes. The authors again used data from previous research to choose members of this group: they relied on two studies which had identified a large number of multiprotein complexes in yeast. Using this group of genes for comparison, the authors found that, like essential genes, genes encoding proteins involved in multiprotein complexes generally display less noise than other genes. This study draws a simple but fundamental conclusion about noise in eukaryotic gene expression—noise has physiological consequences. Importantly, the fact that noise is minimized in those gene groups for which noisy expression would be most harmful suggests that factors contributing to noise are subject to natural selection. This study also demonstrates the power of using the growing number of genomescale datasets in this type of analysis. Researchers will undoubtedly continue to mine the available data to draw biological conclusions not anticipated by the original authors.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC406520.xml

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