Drosophila melanogaster. However, we still know little about the descending neural pathways from the higher order processing centers and how these connect with other neural circuits. Here we describe, in detail, the adult projections patterns that arise from a cluster of 78 neurons, defined by the expression of the Odd-skipped transcription factor. We term these neurons Odd neurons. By using expression of genetically encoded axonal and dendritic markers, we show that a subset of the Odd neurons projects dendrites into the calyx of the mushroom body (MB) and axons into the inferior protocerebrum. We exclude the possibility that the Odd neurons are part of the well-known Kenyon cells whose projections form the MB and conclude that the Odd neurons belong to a previously not described class of extrinsic MB neurons. In addition, three of the Odd neurons project into the lobula plate of the optic lobe, and two of these cells extend axons ipsi- and contralaterally in the brain. Anatomically, these cells do not resemble any previously described lobula plate tangential cells (LPTCs) in Drosophila. We show that the Odd neurons are predominantly cholinergic but also include a small number of γ-aminobutyric acid (GABA)ergic neurons. Finally, we provide evidence that the Odd neurons are a hemilineage, suggesting they are born from a defined set of neuroblasts. Our anatomical analysis hints at the possibility that subgroups of Odd neurons could be involved in olfactory and visual processing.Olfactory processing has been intensively studied in  Drosophila melanogaster brain as there are few morphological landmarks by which to identify specific neural groups. However, the advent of genetic techniques such as the Gal4/UAS system and other binary systems , which project to the antennal lobe (AL) where they synapse on to projection neurons (PNs), thereby forming several glomeruli Stocker, . From thDrosophila requires visual and olfactory integration to track an odor plume effectively , ventromedial protocerebrum (VMPR), and ventrolateral protocerebrum (VLPR). We also identify the different neurotransmitters expressed in the Odd neurons. Using the MARCM   line    to suppress DenMark until adult stages. To address whether Kenyon cells express Odd-skipped, we used a line containing a LacZ insertion in the Odd-skipped locus: Oddrk111 (kind gift of Cordelia Rauskolb)  and UAS-CD8GFP (Bloomington Stock Center). MARCM clones  Odd-Gal4, UAS-CD8GFP and FRT19A,hsFLP,Tub-Gal80 (Bloomington Stock Center). Expression of neurotransmitters was assessed by using the Chat-Gal4 line  crossed to QUAS-mtdTomato-3xHA (Bloomington Stock Center) . It is a mouse monoclonal antibody and recognizes the protein Bruchpilot . The antibody has been used previously against transgenically expressed β-galactosidase in Drosophila  and is a polyclonal chicken antibody raised against the full-length native The anti-FasII antibody is a mouse monoclonal antibody made by DSHB . The antibody was raised against a fusion protein containing the C-terminal 496 amino acids, which includes the cytoplasmic domain. The staining disappears in FasII knock-out flies  is a mouse monoclonal antibody generated against the extracellular domain of the Aequorea victoria. It was purified by ion-exchange and has been used previously in Drosophila to label genetically expressed GFP by comparing localization of expression in specimens with and without GFP expression  is a rabbit polyclonal raised directly against green fluorescent protein (GFP) isolated from Drosophila to detect genetically expressed GFP by comparing localization of expression in specimens with and without GFP expression . They were raised against partially purified recombinant GFP. They detect specifically GFP and GFP-fusion proteins (manufacturer's notes). This antibody has been used previously in Drosophila brains previously . It was raised against a conjugate of γ-aminobutyric acid (GABA)–bovine serum albumin and affinity immunopurified. The antiserum was characterized by dot-blot immunoassays by the manufacturer and has been used to label GABA protein in prospero mRNA localization and other anti-prospero antibodies documented in the literature. . The antibody is mouse monoclonal raised against the prospero amino acids sequence 1,196–1,320 as a GST fusion protein; a hybridoma line was generated. Immunolocalization of this antibody is similar to that described for both Drosophila . It was generated against bacterially expressed full-length protein. Specificity of the antibody was tested by comparing antibody staining between wild-type flies and repo-null mutant flies. This antibody is routinely used in Drosophila to label Repo-expressing cells.  that specifically recognizes the Drosophila choline acetyltransferase and is used as a marker for cholinergic neurons. It is a mouse monoclonal antibody produced by DSHB . Antibody specificity was confirmed by western blotting where the antibody recognizes a 83-kDa band that is the predicted size of the fusion protein used to generate the antibody . In flies where RFP is targeted to epithelial glia using pfFRC71antibody binding is seen in the known expression pattern of pfFRC7. In addition, antibody labeling is absent in flies in which RFP is not expressed  targets Drosophila embryos, which resulted in a similar pattern of Odd-skipped expression as that reported previously in the literature. Furthermore, staining is absent in Odd-skipped null mutant embryos  were: Alexa Fluor 488 donkey anti-rabbit, Alexa Fluor 488 goat anti-mouse, Alexa Fluor 546 donkey anti-chick, Alexa Fluor 546 goat anti-mouse, Alexa Fluor 546 goat-anti-rabbit, and Alexa Fluor 633 donkey anti-rabbit. Secondary antibodies were used at a 1:500 dilution.Adult, pupal, and larval brains were dissected in cold phosphate-buffered saline  and fixed in PBS-buffered 4% formaldehyde for 30 minutes. Brains were washed several times in PBS and stored in methanol at 20°C. Standard antibody labeling was followed  was used instead of Triton X-100. Also primary antibodies were incubated overnight and secondary antibodies for 2 hours.MARCM clones were generated as previously described  originate predominantly from the Johnston organ situated at the second antennal segment. To specifically label these neurons we used DiI  at a working dilution of 6 μg/μl in DMF (dimethylformamide). Adult Odd-gal4 UAS All images were taken by using a Zeiss 510 confocal microscope. Unless otherwise stated, all images were acquired as a z-stack with 2-μm intervals between each confocal section. Images were acquired by using either 20× air or 40× oil immersion objectives at a resolution of 512 × 512 pixels. The degree of further magnification is given in the figure legends. Image J was initially used to generate z-stacks or individual sections of the images acquired. Beyond this there was little image manipulation apart from general adjustment of brightness and contrast in Photoshop .rk111 line, which is an insertion of the LacZ gene in the Odd locus  also overlaps ). A posterior view of the brain shows the entire distribution of the Odd arbor A. The OdDrosophila brain. By quantifying the number of Odd-skipped positive glia, we can deduce that the rest of the Odd cells must be neuronal. To label glia specifically we used an antibody against Reversed Polarity (Repo), a well-known glial marker  , whereas we often saw single-cell clones. This strongly suggests that the Odd neurons are one hemilineage, as we would expect to see two cell clones if both GMC lineages expressed Odd-skipped. Consistent with this we see two types of single-cell clones in the embryo, one produced by each NB. In the larvae only one NB generates Odd neurons and so we only see one type of single-cell clone in each temporal window of clonal induction in the larvae.Taken together, these data strongly support the idea that the Odd neuronal cluster seen in the adult arises from defined NBs. We did not see two cell clones in any of our brains regardless of the developmental stage of clonal induction   , which are sensitive to small moving objects but not background motion  strongly indicates that the Odd neurons are a hemilineage. This would also fit with the number of cells generated during larval and pupal development, which is about 60 cells. These cells are generated over a period of 4 days and considering that an average cell cycle is 1 hour (Campos-Ortega and Hartenstein, The fact that we never see two cell clones (


Combinatorial sensor arrays, such as the olfactory system, can detect a large number of analytes using a relatively small number of receptors. However, the complex pattern of receptor responses to even a single analyte, coupled with the non-linearity of responses to mixtures of analytes, makes quantitative prediction of compound concentrations in a mixture a challenging task. Here we develop a physical model that explicitly takes receptor-ligand interactions into account, and apply it to infer concentrations of highly related sugar nucleotides from the output of four engineered G-protein-coupled receptors. We also derive design principles that enable accurate mixture discrimination with cross-specific sensor arrays. The optimal sensor parameters exhibit relatively weak dependence on component concentrations, making a single designed array useful for analyzing a sizable range of mixtures. The maximum number of mixture components that can be successfully discriminated is twice the number of sensors in the array. Finally, antagonistic receptor responses, well-known to play an important role in natural olfactory systems, prove to be essential for the accurate prediction of component concentrations. in silico designs of receptor arrays reveal that antagonistic responses, in which the receptor is bound by the ligand but there is no downstream reporter activity, are necessary for precise recognition of mixture components. This conclusion provides a rationale for the widespread inhibitory responses observed in olfactory systems. Our methodology can be employed with both biological systems and artificial receptor arrays (“electronic noses”) designed for various industrial needs.Mammalian and insect olfactory systems are combinatorial in nature - instead of activating a single specialized receptor, each analyte invokes a complex pattern of responses across the receptor array. The advantage of such systems lies in their ability to detect a large number of analytes with a relatively small number of receptors. However, the complexity of array responses to mixtures of analytes makes quantitative prediction of component concentrations a challenging task. Here we show that combinatorial output from an array of four engineered G-protein-coupled receptors can be used to predict the concentration of each component in mixtures of highly related sugar nucleotides. We employ a physical model of ligand-receptor interactions and carry out Bayesian analysis of the array output. Furthermore, our  Mammalian and insect olfactory systems are capable of recognizing tens of thousands of odors – mostly organic compounds with diverse chemical structures and properties The idea of combinatorial recognition has been adapted to artificial arrays in which multiple sensors with partially overlapping selectivities respond to a given analyte Here we describe a physical model of receptor-ligand recognition that explicitly relates observed response patterns to component concentrations and receptor properties, making it easier to quantify mixture constituents. We use Bayesian inference to predict absolute concentrations of each ligand in arbitrary mixtures of uridine diphosphate (UDP) sugar nucleotides applied to a combinatorial array of four GPCRs. Furthermore, we develop a universal metric of receptor array performance, and use it to study the fundamental limits imposed on the accuracy of ligand recognition by the physics and biology of receptor-ligand interactions. Finally, we provide design guidelines for constructing cross-specific arrays optimized for mixture recognition, and demonstrate that inhibitory responses are essential for simultaneous detection of all components in a complex mixture.in vitro from the human UDP-glucose receptor using directed mutagenesis of the residues involved in ligand binding  S.cerevisiae. To do so, we replaced the yeast pheromone receptor with one of the sensor GPCRs in strains in which the pheromone response pathway was modified to respond to the heterologous receptor by inducing transcription of the E. coli lacZ gene Materials and Methods). Applying a mixture of nucleotide sugars to the receptor array yields a complex pattern of responses of the four receptor-bearing strains. The response of each receptor depends on the concentration of all components in the mixture, on the receptor-ligand binding affinities, and on the efficacy with which each ligand activates the receptor. Nonetheless, the contents of arbitrary nucleotide sugar mixtures can be deciphered using array readout as input to a physical model of receptor-ligand interactions.Our sensor array is comprised of four engineered receptors  with distinct but overlapping specificities for four types of nucleotide sugars: UDP-glucose (UDP-Glc), UDP-galactose , UDP-glucosamine (UDP-GlcNAc) and UDP. The receptors were evolved Materials and Methods). Reporter activation measurements as a function of single ligand concentration are shown in Fig. S1a. We use these data to estimate the parameters of Eq. (1) . The most likely values of the parameters (Table S1) are then used for subsequent evaluation of mixtures of compounds. The accuracy of parameter predictions depends on the range of concentrations available for these calibration experiments  and on the amount of experimental noise .We start with the simplest case in which a receptor interacts with a single ligand. We assume that the observed signal in our receptor-bearing reporter strain is proportional to the probability that the receptor is bound by the ligand. This proportionality value,  Eq. (1) Fig. 1a Table S1). Assuming that all ligands bind competitively to the same site on the receptor, we model the response of the receptor-bearing strain to mixtures of compounds by calculating the total intensity as a sum of fractional occupancies of the receptor by each ligand weighted by the corresponding efficacies (Eq. (6)). We treat each of the receptor-bearing strains with an unknown mixture, sequentially diluted to provide a series of samples across a million-fold range of concentrations . We carry out Bayesian inference for the entire receptor array, predicting the total concentration of all ligands and the concentration ratios of ligand pairs . As before, we use a four-ligand model, which should predict zero concentrations for UDP-GlcNAc and UDP. The predicted values of Table S4), showing that our approach is not overly sensitive to the arbitrary definition of relative concentrations.Our second test involved combining UDP-Glc and UDP-Gal in several unequal proportions and applying the resulting mixture to the four-receptor array . As expected, the errors rapidly get smaller as the number of receptors is increased, making larger arrays unnecessary. Surprisingly, in several cases adding extra receptors makes the errors somewhat worse before they become better again , indicating that the noise in the new data outweighs the benefit of additional measurements.Increasing the number of receptors should improve prediction accuracy by providing additional information about the mixture. To see the extent of these improvements, we have used a variable number of receptors to infer component concentrations in six equal-proportion mixtures of two nucleotide sugars from Fig. S1a), the receptors differ from each other in fairly subtle ways. In particular, different ligands do not invoke markedly orthogonal profiles of receptor responses. Nonetheless, even with this suboptimal array design, our algorithm provides accurate identification of ligands present in a mixture and a reasonable assessment of the relative amounts of each.As evident from the activation profile of each receptor in response to each ligand ), which can be visualized as a multidimensional landscape. The global maximum on this landscape corresponds to the model that best describes the data, while the curvature at the maximum shows how sensitive the likelihood is to the change in each parameter. Narrow peaks result in precisely defined parameter values, whereas wide plateaus yield many nearly equivalent predictions and therefore sizable uncertainties in parameter estimates. Expanding the log-likelihood in the vicinity of its maximum yields a Hessian matrix (Eq. (9)), which contains information about standard deviation Table S1). We observe close correspondence between parameter uncertainties inferred from nested sampling vs. Hessian analysis . Moreover, since larger uncertainties make it easier for the average values of predicted parameters to be incorrect, there is also correlation between Hessian errors and the absolute differences between mean predicted and true values . The Hessian-based approach remains useful when experimental data, for which the precise model is unknown and the noise is substantially higher (Table S1), is analyzed in the same way: the average over Table S1. We conclude that Hessian errors are a reasonable measure of sensor array performance.Hessian analysis relies on the quadratic expansion in the vicinity of the log-likelihood maximum and hence it is important to check how well it captures the behavior of the more general but computationally intensive nested sampling approach. To create a test case for which the answer is known, we have used Eq. (6) to generate synthetic data for 15 equal-proportion mixtures from Not all receptors are equally good candidates for inclusion into biosensor arrays – for example, receptors with similar sets of efficacies and binding affinities should be less useful than receptors with more orthogonal binding and activation patterns. Here we make such qualitative insights precise by developing a Hessian approach to biosensor array design. That is, given a certain number of measurements with an array of fixed size , we wish to derive the most optimal choice of receptor properties for deciphering the mixture. From the Hessian point of view, the best array will have the smallest errors in predicting component concentrations (Eq. (10)). Because each error is inversely proportional to the determinant of the Hessian, we maximize the determinant instead of minimizing the errors directly. Similarly to the prediction of constituent concentrations, the maximization is carried out by nested sampling orFig. S7). The actual values of the binding energies depend on the relative concentration To demonstrate our approach, we first optimize parameters of a single receptor discriminating a mixture of two ligands. By maximizing the determinant of the Hessian, in this case a Fig. S7 and S8). The single-peak landscape structure appears only if the absolute concentrations of both components need to be predicted together. Strikingly, simultaneous prediction of the total and relative concentrations is impossible with the agonist-agonist receptor response . The dependence of the optimal binding energies on the value of Fig. S10). Thus one set of The fine-tuning of binding energies is not necessary if either the total concentration The agonist-antagonist pattern observed in the one-receptor, two-ligand case plays the role of a basic building block when two or more receptors interact with multiple ligands: nested sampling maximization of the Hessian determinant with respect to binding energies Fig. S4). If we eliminate the agonist-antagonist degree of freedom by setting all efficacies to In the light of the observed agonist-antagonist behavior, it is not surprising to see that each receptor can identify concentrations of at most two ligands (Text S1 for a complete enumeration).The patterns shown in In general, Fig. S11a). This intensity modulation provides enough information for decoding the contents of the mixture. Similarly, in the two-receptor, three-ligand case . This pattern is distinct from those induced by single ligands and by binary mixtures with the same relative concentrations as in the three-ligand case.The agonist-antagonist rules described above create readout patterns that are not a simple sum of array responses to single-ligand binding. For one receptor optimized to discriminate two ligands . In particular, if Fig. S12c). Conversely, if Fig. S12d). Not surprisingly, the agonist-antagonist patterns are even more pronounced if both Fig. S12e and S12f). Because two and certainly three optimized receptors are sufficient for discriminating four-ligand mixtures . This observation is consistent with Hessian analysis: for example, the average Hessian uncertainties for three UDP-free binary mixtures are For the experimentally implemented four-receptor GPCR array, nested sampling errors are consistently larger when UDP is present in the mixture .For each single ligand or combination of ligands, a series of measurements was performed at several values of the total concentration For a single receptor interacting with a single ligand, we model the normalized reporter fluorescent intensity as:We compute the log-likelihood of the data by assuming that fluorescence measurements are Gaussian-distributed around values from Eq. (1):The reporter response to a mixture of ligands is given byThe log-likelihood of the observed pattern of fluorescence intensities from multiple receptors interrogated by a mixture of ligands is given byWe estimate all posterior probabilities by nested sampling Text S1):http://olfaction.rutgers.edu.The Hessian matrix in the low-noise limit can be written as  Each receptor-ligand combination is tested for functional activation, yielding 16 binding curves. For each curve, intensity normalized by the maximum intensity on the plate is plotted against b) An unknown mixture of four ligands is applied to each of the four receptors. The resulting fluorescent response curves together with the (EPS)Click here for additional data file.Figure S2Prediction accuracy increases with the number of measurements at different total concentrations. Synthetic data was generated using Eq. (1) in the main text, with (EPS)Click here for additional data file.Figure S3Prediction accuracy decreases with the amount of noise in the data. Synthetic data was generated using Eq. (1) in the main text, with (EPS)Click here for additional data file.Figure S4Inference of ligand concentrations is improved with the number of receptors interrogating the mixture. Shown on the log-scale are means and standard deviations for three relative concentrations Click here for additional data file.Figure S5Hessian uncertainties vs. standard deviations from nested sampling. Synthetic data was generated as four replicates for each of the 15 equal-proportion mixtures from Table S1 and Materials and Methods). For each parameter, a Hessian error computed using Eq. (10) in the main text (x-axis) is compared with the standard deviation from a nested sampling run (y-axis). Nested sampling simultaneously infers relative concentrations Table S1.(EPS)Click here for additional data file.Figure S6Hessian uncertainties vs. errors in parameter predictions. Synthetic data was generated as four replicates for each of the 15 equal-proportion mixtures from Table S1 and Materials and Methods). For each parameter, a Hessian error computed using Eq. (10) in the main text (x-axis) is compared with the absolute magnitude of the difference between the mean value predicted by nested sampling and the correct value (y-axis). Nested sampling simultaneously infers relative concentrations Table S1.(EPS)Click here for additional data file.Figure S7Matrix elements and the determinant of the Hessian in the antagonist-agonist case, plotted as a function of binding energies  The efficacies are fixed at (EPS)Click here for additional data file.Figure S8Matrix elements of the Hessian in the agonist-antagonist case, plotted as a function of binding energies  The efficacies are fixed at (EPS)Click here for additional data file.Figure S9Matrix elements and the determinant of the Hessian in the agonist-agonist case, plotted as a function of binding energies  The efficacies are fixed at (EPS)Click here for additional data file.Figure S10Changes in the Hessian determinant with the concentration of the second ligand. Each determinant is plotted as a function of binding energies (EPS)Click here for additional data file.Figure S11Schematic diagram of receptor activation by single ligands and ligand mixtures with optimized binding affinities and efficacies. (a) Two cases of agonist-antagonist single-receptor arrays designed to discriminate a mixture of two ligands (b) Array of two receptors designed to discriminate a mixture of three ligands ((EPS)Click here for additional data file.Figure S12Improving performance of the experimentally implemented sensor array. Free energies a) and efficacies b) in the experimentally implemented sensor array with parameters from Table S1. Free energies c) and efficacies d) from two sensor arrays in which the determinant of the Hessian was maximized only with respect to e) and efficacies f) in the optimal sensor array in which the determinant of the Hessian was maximized with respect to both Materials and Methods). In panels a–d, the order of ligands is L1: UDP, L2: UDP-Gal, L3: UDP-Glc, L4: UDP-GlcNAc. The order of receptors is R1: H-20, R2: K-3, R3: L-3, R4: 2211. Note that ligand and receptor identities are lost in panels e,f since all parameters have been optimized.(EPS)Click here for additional data file.Dataset S1Normalized fluorescence intensity measurements from experiments with equal-proportion  and unequal-proportion  mixtures.(XLS)Click here for additional data file.Table S1Parameters of receptor-ligand interactions predicted from one-receptor, one-ligand binding curves  and one-receptor, two-ligand binding curves (UDP).(PDF)Click here for additional data file.Table S2Prediction of ligand concentrations in equal-proportion mixtures (data for ).(PDF)Click here for additional data file.Table S3Prediction of ligand concentrations in unequal-proportion binary mixtures of [UDP-Gal] and [UDP-Glc] (data for ).(PDF)Click here for additional data file.Table S4Prediction of ligand concentrations in unequal-proportion binary mixtures of [UDP-Gal] and [UDP-Glc] using an alternative definition of relative concentrations. We used nested sampling of a four-receptor, four-ligand model to infer relative concentrations Table S1 and used everywhere else) between different plates. For Plate 1 (measurements 1–3), Table S1.(PDF)Click here for additional data file.Text S1Supplementary Materials and Methods.(PDF)Click here for additional data file.


Cervical cancer is the third most common cancer among women worldwide, and in Nigeria it is the second most common female cancer. Cervical cancer is an AIDS-defining cancer; however, HIV only marginally increases the risk of cervical pre-cancer and cancer. In this study, we examine the risk factors for cervical pre-cancer and cancer among HIV-positive women screened for cervical cancer at two medical institutions in Abuja, Nigeria.A total of 2,501 HIV-positive women participating in the cervical cancer screen-and-treat program in Abuja, Nigeria consented to this study and provided socio-demographic and clinical information. Log-binomial models were used to calculate relative risk (RR) and 95% confidence intervals (95%CI) for the risk factors of cervical pre-cancer and cancer.3 or more also had lower risk of screening positivity or invasive cancer diagnosis . Other factors such as having had 5 or more abortions  and the presence of other vaginal wall abnormalities  were associated with screening positivity or invasive cancer diagnosis.There was a 6% prevalence of cervical pre-cancer and cancer in the study population of HIV-positive women. The risk of screening positivity or invasive cancer diagnosis reduced with increasing age, with women aged 40 years and older having the lowest risk . Women with a CD4 count of 650 per mmThe prevalence of screening positive lesions or cervical cancer was lower than most previous reports from Africa. HIV-positive Nigerian women were at a marginally increased risk of cervical pre-cancer and cancer. These findings highlight the need for more epidemiological studies of cervical cancer and pre-cancerous lesions among HIV-positive women in Africa and an improved understanding of incidence and risk factors. In 2008, cervical cancer was the third most common cancer among women and the seventh most common cancer overall with 530,000 new cases and 275,000 deaths reported . Women iVirtually all cases of cervical pre-cancer and cancer are associated with a high-risk human papillomavirus (hrHPV) infection, with types 16 and 18 reported to account for the majority of cases ,5. HowevSince the onset of the HIV epidemic, the United States Centers for Disease Control and Prevention (CDC) has classified cervical cancer, Kaposi sarcoma and non-Hodgkin’s lymphoma as AIDS-defining cancers because of their close association with HIV infection . HoweverIn developed countries, some 75% of women are screened—typically by Papanicolaou staining (Pap smears) and more recently by human papillomavirus (HPV) DNA-based tests—compared with 5% in developing countries . As a coAn alternative cervical cancer screening method that is recommended for use in developing countries is visual inspection with acetic acid or Lugol’s iodine (VIA/VILI). VIA/VILI is an inexpensive test that can be easily adopted and scaled up in the healthcare system of LMIC, does not require specialized laboratories or equipment, can be performed by a wide range of health care professionals after a short period of training and does not require several visits by patients ,16,19,22In some countries, donor funding from the President’s Emergency Plan for AIDS Relief (PEPFAR) and the Global Fund has provided the resources and infrastructure needed to scale up visual inspection with acetic acid testing (VIA) . For exaThis study was carried out at the University of Abuja Teaching Hospital and the National Hospital, both in Abuja, Nigeria.HIV-positive women older than 18 years of age either on anti-retroviral therapy (ART) or ART naïve attending PEPFAR treatment clinics for HIV care were enrolled at both sites. Participants were women who voluntarily presented and gave informed consent for cervical cancer screening.After obtaining informed consent, nurses obtained demographic and clinical information on all participants and examined the introitus and vulva, noting any abnormalities. A bivalve speculum was then introduced into the vagina for examination of the cervix using a halogen lamp. The squamo-columnar junction of the cervix (transformation zone) was identified and any secretions or exudate cleaned off before a nurse applied a cotton wool swab soaked in 5% acetic acid solution for 3 minutes. The results of each examination were noted. Pre-cancerous lesions were defined as being dense aceto-white lesions with well-defined margins observed within the vicinity of the transformation zone originating from the squamo-columnar junction, or if the whole cervix or cervical growth turned white. A suspicion of cancer was defined as any cervical ulcer or growth being observed . FollowiResults of VIA or VILI were classified according to the International Agency for Research Against Cancer (IARC) manual and recorded after each test . PositivWomen with confirmed VIA or VILI positive results were offered immediate treatment with cold coagulation. This is an ablative therapy used for treatment of intraepithelial neoplasia that is not suspected of being cancerous, not covering more than 75% of the cervix, is not disappearing into the endo-cervical canal, and the squamo-columnar junction is fully visible. A patient with a cervix suspicious for cancer or with intraepithelial neoplasia was ineligible for cold coagulation according to predefined criteria. These women were referred to the gynecology clinic to undergo colposcopy and biopsy to confirm probable cancer diagnosis and to receive further treatment, as is standard practice in screen and treat programs .At the end of every week, quality assurance meetings were held with nurses and the consulting gynecologist to review all cervigrams taken during the week, with the aim of assessing the nurses’ visual detection skills and referral decisions. In those cases where there was a missed diagnosis of clinical relevance the patient was recalled.Relative risk and 95% confidence intervals (95%CI) were calculated using log-binomial regression models ,26. The 3. The majority of the HIV-infected women, 86%, were asymptomatic and were classified as having Stage 1 at baseline as defined by World Health Organization guidelines [Socio-demographic and clinical characteristics of the study participants are shown in Tables idelines .trend < 0.0001). The median number of pregnancies among women with a positive VIA result/invasive cancer was 3 compared with 4 pregnancies in women with a negative (ptrend=0.03). A greater proportion of women with VIA positive lesions were Christians (p=0.04). Women with a positive screening result/invasive cancer also had a higher proportion of vaginal wall abnormality (p=0.0006). The most recent CD4 count prior to screening was significantly higher among women with a negative screening result compared with women with a positive screening result/invasive cancer (p <0.0001). The results of the multivariate analyses are shown in Table 3 or more had the lowest risk of screening positivity or invasive cancer diagnosis . Other independent risk factors, such as having had five or more abortions  and the presence of other vaginal wall abnormalities , were associated with screening positivity or invasive cancer diagnosis. There were no independent associations with age at first sexual intercourse or multiple pregnancies. We detected non-linearity in the relationship between age and screening status, where risk was slightly elevated up to age 35 and then declined dramatically up to age 50, after which no further decrease in risk was apparent , and the median age observed for an invasive cancer diagnosis or positive screening result was 32 years, which is about 10 years younger than the median age of 47 years for cervical cancer diagnosis in the general population . This re3 had low incidence of cervical pre-cancer compared with those with lower CD4 counts [The results of this study suggest that HIV-infected women with higher CD4 cell counts were at a reduced risk for invasive cancer diagnosis or a positive VIA result. This result agrees with findings from other studies. For example, in Rwanda, an inverse association was found between the CD4 count and cervical pre-cancer among HIV-infected women . Harris 4 counts . These f4 counts .We also found the risk for cervical pre-cancer and cancer to be associated with age (≥40 years) which supports the natural history of cervical carcinogenesis from hrHPV infection through cervical dysplasia, pre-cancer and cancer; having five or more abortions and the presence of vaginal wall abnormalities suggesting a generalized increased risk of sexually transmitted diseases in women who have cervical pre-cancer and cancer. The non-linearity in the relationship between age and screening status may be due to the natural history of cervical carcinogenesis where infection needs to persist before engendering malignant change .As this was a cross-sectional study it was not possible to explore cause-and-effect relationships. We did not perform HPV DNA testing, cytology or biopsies to confirm the result of visual inspections; as a result, visually inapparent pre-cancerous lesions could have been missed. Also, since this study was conducted among HIV-infected women on ART, the results may not be generalizable to uninfected women. Nevertheless, we believe this study demonstrates the risk factors of positive cervical cancer screening test in this population.In conclusion, HIV-positive Nigerian women are at a marginally increased risk for cervical pre-cancer and cancer. Our study highlight the importance of screening HIV-positive women in Nigeria for cervical cancer. The screen and treat approach using VIA/VILI has been shown to be effective for the detection of cervical pre-cancer and cancer, and incorporation of immediate treatment where applicable is associated with reduced loss to follow-up. This can also be rapidly scaled up in resource-constrained settings and be incorporated into HIV treatment programs. Our results demonstrate the need for more epidemiological studies of cervical pre-cancer and cancer among HIV-positive, indeed all women in Africa and an improved understanding of incidence and risk factors.Written informed consent was obtained from the patient for publication of this report and any accompanying images.The authors declare that they have no competing interests.UO conducted the data analysis and drafted the manuscript. MA implemented the study, collected data, assisted with data analysis and drafting of the manuscript. ZM implemented the study, collected data, assisted with data analysis and drafting of the manuscript. IL worked on study implementation and data collection. RO supervised clinical data collection and assisted with drafting the manuscript. OO supervised clinical data collection and assisted with drafting the manuscript. PD supervised clinical data collection and assisted with drafting the manuscript. DS supervised data analysis and writing of the manuscript. WB supervised clinical data collection and assisted with drafting the manuscript. CA conceived the study, participated in study implementation, data collection, data analysis and drafting of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/582/prepub


Antiretroviral therapy (ART) has been remarkably effective in ameliorating Human Immunodeficiency Virus (HIV)-associated morbidity and mortality. The rapid decline in viral load during ART also presents an opportunity to develop a “treatment as prevention” strategy in order to reduce HIV transmission at a population level. Modelling exercises have demonstrated that for this strategy to be effective, early initiation of ART with high coverage of the HIV-infected population will be required. The HIV epidemic has fueled a resurgence of tuberculosis (TB) particularly in sub-Saharan Africa and widespread early initiation of ART could also impact this epidemic via several mechanisms. The proportion of patients with low CD4 cell counts who are at high risk of TB disease from progression of both latent and new TB infection would be greatly reduced. Entry into a life-long ART program provides an ongoing opportunity for intensified TB case finding among the HIV-infected population. Regular screening for HIV infection also presents an opportunity for intensified TB case finding in the general population. The combined effect of reduced progression of infection to disease and intensified case finding could reduce the overall prevalence of infectious TB, thereby further decreasing TB transmission. In addition, decreasing prevalence of HIV infection would reduce the TB-susceptible pool within the population. The ‘test and treat’ strategy therefore has potential to reduce the TB risk at both an individual and a population level. In this paper we explore the expected “TB dividend” of wider access to ART and also explore the potential of the “test and treat” strategy to impact on TB transmission, particularly in the heavily burdened setting of sub-Saharan Africa. The strong association between Human Immunodeficiency Virus (HIV) and increased risk of tuberculosis (TB) was first described in the mid 1980s in the United States (US) . Soon thIn contrast to the focal HIV-associated TB outbreaks and epidemics in the US, which have been amenable to control with traditional TB control interventions, TB has proved to be a far greater challenge in countries with generalized HIV epidemics . In sub-ART is one of the most potent tools for preventing HIV-associated TB , 11 and 1). Infection may either progress directly to disease (exogenous TB disease) or enter a period of clinical latency before reactivation at a later stage (endogenous TB disease). Like all other infectious diseases, the primary event determining the disease epidemiology is the acquisition of infection with Mycobacterium tuberculosis by a susceptible individual.The pathway whereby HIV-infected individuals transition from the TB-susceptible state to TB infection, TB clinical disease and ultimately to TB treatment and cure is outlined in Fig.  individuals become infected each year and provides an assessment of the current prevailing infection risk in communities with a generalized TB epidemic.In contrast, the mean annual risk of TB infection (ARTI) is calculated from an observed prevalence of infection at a given age and represents the average annual risk of infection since birth that would produce this prevalence. Thus, force of infection represents current infection risk whereas ARTI is an index of average infection risk over time.The mean ARTI is the term most often used to describe risk of acquiring TB infection in childhood and has been used to estimate secular trends in transmission within populations . In deve2) illustrates the impact of high ARTI and high force of TB infection on patients presenting to an ART clinic in scenarios of low, medium and high TB transmission. Latent infection would be present in approximately 1.3%, 12.4% and 74% in settings where ARTI is 0.04%, 0.4%, or 4% per annum, respectively. Active TB disease in the low and medium transmission settings would be predominantly due to reactivation from latent infection, although limited explosive outbreaks may still occur when the local force of infection is high due to uneven distribution within the population [Fig.  in a community and the number of secondary cases resulting from each infectious case (effective contact number). However, the prevalence of TB is also a parameter which is difficult to measure directly and therefore is frequently estimated from other TB parameters such as case notification rates and TB mortality rates . Some daIn scenarios where most TB cases eventually enter the TB control program and in a stable epidemic situation, the mean duration of infectiousness before starting effective chemotherapy can be approximated by the untreated TB prevalence divided by the TB incidence rate. Aggressive scale-up of HIV testing and provision of longitudinal HIV care in communities would provide the opportunity for greatly improved TB case finding, thereby reducing the prevalence of untreated TB. This, in turn, would be associated with shortening of the disease duration and reduced TB transmission risk. Thus, to better understand the potential impact of ART on TB transmission within communities, it will be important to assess the impact of ART scale-up on TB prevalence and disease duration.The impact of social factors on TB transmission and disease has been long recognised. Social crowding in poorly ventilated housing resulted in exceptionally high TB rates in urbanised populations of the nineteenth century . An effeIn Cape Town, the effective contact number is between 11and 22 cases infected for each smear-positive notified case . The effCombination anti-tuberculosis treatment rapidly decreases the infectiousness of drug-sensitive TB cases . TreatmeThe focus of DOTS is on effective case management of self-referred symptomatic patients (passive case finding) and thus removing them from the infective pool. The rates of TB disease are now so low in many industrialised countries that alternative active case finding strategies are no longer considered cost-effective . It has However, in countries with generalized TB and HIV epidemics, the DOTS strategy has failed to reverse the increasing TB burden. In highly burdened countries, many individuals with HIV-associated TB progress to death without their TB disease being recognised -31. WherHaving described how DOTS is insufficient to address the challenge presented by the HIV-associated TB epidemic in Africa, we now consider the potential role of ART, drawing together data on the burden of TB occurring prior to ART, changing TB risk during in cohorts receiving ART and the modelled and observed impact of ART at a population level.South Africa is a country where both HIV and TB are endemic . TB and In addition to those HIV-infected patients referred to the ART clinic with a known TB diagnosis, many of the remaining patients accessing the ART service from other referral sources also had TB diagnosed during intensive pre-ART screening (approximately 25%) , 38. Theex vivo with mycobacterial antigens [in vivo mycobacterial growth [Initiation of ART leads to rapid functional recovery of mycobacterium-specific immune responses, with increasing lymphocyte proliferation of cells which are able to secrete interferon-gamma when stimulated antigens -43. Suchl growth . ART hasl growth .3), use of ART was associated with a TB risk reduction of approximately 80% in patients followed up for 15 months [3 and WHO stage 3 or 4 disease, respectively [In the high burden setting of South Africa . A summary analysis of these results [Data from multiple other cohort studies in both high-income and resource-limited settings report , 45-52 r results  estimate results , suggest3 to >500 cells/ mm3 . It is still unclear whether incident TB during ART is due to predominantly reactivation or re-infection. However, it has been suggested that in the context of a rising CD4 cell count during ART, reinfection is likely to be the predominant mechanism, whereas both are likely to be important in patients with falling CD4 cell counts [TB incidence rates continue to decline during ongoing ART, as a result of ongoing immune recovery -57. The l counts .3); an intermediate risk when CD4 cell counts were in the intermediate range of 200-500 cells/ mm3 and the lowest incidence rates when the current CD4 cell counts exceeded 500 cells/ mm3  [In the Cape Town treatment cohort, the highest TB rates are seen when significant person-time was spent at low CD4 cell counts  . However , 58.4B) demonstrate that the mortality risk reduction associated with current ART was between 64-95% [Case fatality rates are several-fold higher among HIV-infected TB patients than among those without HIV infection, and mortality is related to the degree of immunodeficiency , 60. Anan 64-95% -67. Furtn 64-95%  and a den 64-95% .Current international guidelines recommend ART for all TB co-infected patients regardless of CD4 cell count and it is to be hoped will lead to reductions in TB case fatality rates . However1). In industrialized countries, TB is a rare disease characterised by stochastically-driven small outbreaks rather than by a generalised transmission within the population as a whole [The impact of ART on TB incidence rates in any given population will depend on its impact on the probabilities of transition from TB susceptibility to TB disease and eventual cure, remission or death  [The results of two cross-sectional TB prevalence surveys performed in the same South African community in 2005 and 2008 at different stages of ART scale-up are shown in Fig. (6) , 77. TheThere has been a call to test the efficacy of the “test and treat” strategy in controlled environments . While s1. These represent the parameters with which the impact of implementation of the “test and treat” strategy might be measured. Such evaluation would require prospective surveillance and detailed and comprehensive surveys within implementing communities.long-term HIV prevalence, additional potential impacts on TB epidemic parameters are outlined in Table 3 have a comparable risk to this [5) suggests that it would require greater than 5 years to achieve substantial decreases in the relative risk of incident TB.The primary effects of early and widespread introduction of ART on TB would be by increasing the overall distribution of CD4 cell counts in the HIV-infected population pool. TB risk approximately doubles soon after HIV seroconversion  and pati to this . Thus, pPatients receiving ART have greatly improved survival and will remain at increased risk of incident TB. Net reduction in HIV-associated TB rates will be achieved by reduction of time spent at low CD4 cell counts and further impacted by long-term reductions in HIV prevalence. Occurrence of HIV-associated TB at higher CD4 cell counts overall will be associated with lower mortality risk.The combination of reduced progression from TB infection to TB disease among HIV-infected patients, improved TB case finding among those on ART and incorporation of regular TB symptom screening in the HIV-uninfected population could reduce TB force of infection. Thus, widespread implementation of “test and treat” could use “treatment as prevention” to assist in the control of both the HIV and TB epidemics.There is considerable evidence from observational cohorts that ART improves survival and decreases the rate of progression from both recent and latent TB infection to clinical disease. The population impact of ART is less clear, as increased survival of individuals with low CD4 cell counts could increase the proportion of a population at increased risk of progression to TB disease. In the absence of empiric data, modelling studies have indicated that earlier initiation of ART on a wide scale could significantly impact on TB rates in heavily burdened countries. There is now some early observational data from population studies confirming that TB prevalence and incidence may be reduced by ART.The population impact of ART on HIV-TB rates may occur by three predominant means. First, ART can improve the CD4 cell count profile of a population and therefore decrease progression from infection to TB disease. Second, HIV-infected individuals on ART receive longitudinal care, providing the opportunity for improved TB case detection and decrease in the prevalence of untreated TB. Third, widespread early implementation of ART may reduce the HIV incidence rate and eventually the prevalence of HIV infection.Modelling studies have not explored the TB transmission between the HIV-infected and -uninfected population pools as it has been thought that few HIV negatives acquire TB infection from patients with HIV-associated TB. This interaction may have been underestimated because of the long inter-generation time between transmission of infection from HIV-associated TB cases and subsequent development of TB cases in HIV-negative individuals. Further long-term molecular epidemiologic studies will be required to clarify this interaction. Long-term benefits of HIV-TB control for HIV negatives would be an additional impetus for the “test and treat” strategy.HIV has been considered the primary driver of the explosion of HIV-associated TB epidemic in southern Africa, but it may also be considered to be fuel poured on an existing fire. HIV infection has amplified the high force of TB infection still existing in many parts of the developing world. Control of HIV-associated TB is therefore not isolated from general population TB control. A “test and treat” strategy may provide an additional opportunity for improved general population screening for TB in high burdened settings.


These findings have significant implications for graft adaptation and long-term patency.It is well known that flow patterns at the anastomosis of coronary artery bypass graft (CABG) are complex and may affect the long-term patency. Various attempts at optimal designs of anastomosis have not improved long-term patency. Here, we hypothesize that mild anastomotic stenosis (area stenosis of about 40–60%) may be adaptive to enhance the hemodynamic conditions, which may contribute to slower progression of atherosclerosis. We further hypothesize that proximal/distal sites to the stenosis have converse changes that may be a risk factor for the diffuse expansion of atherosclerosis from the site of stenosis. Twelve (12) patient-specific models with various stenotic degrees were extracted from computed tomography images using a validated segmentation software package. A 3-D finite element model was used to compute flow patterns including wall shear stress (WSS) and its spatial and temporal gradients . The flow simulations showed that mild anastomotic stenosis significantly increased WSS (>15 dynes⋅cm Coronary artery bypass graft (CABG) is a highly-effective therapeutic treatment to relieve symptoms of ischemic heart diseases Computational fluid dynamics (CFD) is a common method to determine hemodynamic risk factors  for atherosclerosis In the present study, a transient 3-D finite element (FE) model was used to solve the continuity and Navier-Stokes equations with the measured inlet flow boundary condition and the stress-free outlet boundary condition in geometrical models of SVG and coronary arteries obtained from patient computer tomography (CT) images. The hemodynamic parameters including WSS, OSI, and WSSG were computed at anastomoses based on the computed flow field. These parameters were also computed in four idealized geometric models to mimic the potential effects of various SVG anastomoses on flow patterns, which were compared with those in patient-specific SVG models to show the effects of mild stenosis on local flow dynamics. The significance, implication and limitation of flow simulations are discussed in relation to the long-term SVG patency.The purpose of this retrospective study was to investigate hemodynamic changes near anastomoses of SVG and coronary artery that had stenoses of various degrees. Twelve human subjects underwent CT angiography (CTA) of coronary arteries for >1 year after the SVG connected the aorta to the coronary artery with end-to-side anastomosis. The retrospective study was approved by the Institutional Review Board (IRB) for Indiana University and the human subjects provided written informed consent to participate in this study. A flow chart in Prior to the imaging acquisition, patients were given repeated doses of intravenous metoprolol of 5 mg every 5 minutes until heart rate was ≤65 bpm or a maximum dose of 15 mg was given. All patients received sublingual nitroglycerin tablet (0.4 mg) 3–5 minutes before CT examination.All studies were performed on a dual-source CT scanner  similar to a previous study As shown in P, ρ, and μ represent velocity, pressure, blood mass density, and viscosity, respectively The governing equations are formulated for SVG and coronary artery, each vessel of which is assumed to be rigid and to have impermeable wall. The equations of continuity and Navier-Stokes can be written as:μ) and density (ρ) of the solution were assumed as 4.0 cp and 1.06 g/cm3, respectively, to mimic blood flow with a hematocrit of about 45% in these arteries.A FORTRAN program was used to implement the Galerkin FE method, which has been validated by theoretical solutions and experimental measurements To evaluate the relation between graft angle and graft-to-host cross-sectional area (CSA) ratio, we also constructed four idealized geometric models , OSI, and time-averaged velocity (Unit: cm/s) at anastomoses with small and mild stenoses, respectively.−2) at the anastomosis between SVG and coronary artery for the four idealized models. −2) and OSI, respectively, in Region A , where a decrease of graft-to-host CSA ratio (−3) in Regions B and D, respectively, where a decrease of graft-to-host CSA ratio as well as an increase of graft angle  enhances hemodynamic conditions  albeit the proximal/distal sites to the stenosis show deteriorated hemodynamics. This suggests retarded atherosclerosis inside the stenosis, with enhanced atherosclerosis upstream and downstream of stenosis.The reconstructed anastomoses showed mild stenoses in most patients for ≥10 years after operation, but relatively less in patients for >1 year and <6 years. These anastomotic stenoses were mainly attributed to atherosclerosis −2) and high OSI (≥0.15) at toe and heal regions of small anastomotic stenosis between SVG and coronary artery, as shown in −2) inside the stenosis given that −2), but no significant increase of OSI at proximal and distal sites to the mild stenosis, as shown in There were low time-averaged WSS  Moreover, the decrease in WSS at proximal and distal sites to the stenosis is mainly caused by the stagnated flows, as shown in The effect of geometry of anastomosis on arterial bypass graft patency has been investigated by many researchers In comparison with the anastomosis in −2) and decreases OSI (<0.02) and results in more uniform distribution of hemodynamic parameters inside the anastomosis in An increase of graft-to-host CSA ratio or a decrease of graft angle improves hemodynamic conditions at the anastomosis between SVG and coronary artery which is consistent with previous studies The flow velocity at the inlet of SVG was obtained from literature measurements Patient-specific mild anastomotic stenosis (area stenosis of 40–60%) increased WSS and decreased OSI inside the stenosis, but had a converse effect on WSS at proximal and distal sites to the stenosis. The favorable hemodynamics inside a mild stenosis may be a stabilizing factor to decrease the rate of graft failure despite the initial designs with no optimal graft-to-host CSA ratios or graft angles. Moreover, the changes at proximal and distal sites to the stenosis may contribute to diffuse expansion of atherosclerosis from a mild stenosis which could ultimately affect the long-term graft patency. To bolster the patient-specific conclusions, we also carried out the flow simulations in four idealized types of anastomoses and found that an increase of graft-to-host CSA ratio or a decrease of graft angle, between SVG and coronary artery, improved hemodynamic conditions.Appendix S1Appendix.(DOCX)Click here for additional data file.


Past studies of the human intestinal microbiota are potentially confounded by the common practice of using bowel-cleansing preparations. We examined if colonic lavage changes the natural state of enteric mucosal-adherent microbes in healthy human subjects.Twelve healthy individuals were divided into three groups; experimental group, control group one, and control group two. Subjects in the experimental group underwent an un-prepped flexible sigmoidoscopy with biopsies. Within two weeks, subjects were given a standard polyethylene glycol-based bowel cleansing preparation followed by a second flexible sigmoidoscopy. Subjects in control group one underwent two un-prepped flexible sigmoidoscopies within one week. Subjects in the second control group underwent an un-prepped flexible sigmoidoscopy followed by a second flexible sigmoidoscopy after a 24-hour clear liquid diet within one week. The mucosa-associated microbial communities from the two procedures in each subject were compared using 16S rRNA gene based terminal restriction fragment length polymorphism (T-RFLP), and library cloning and sequencing.Clone library sequencing analysis showed that there were changes in the composition of the mucosa-associated microbiota in subjects after colonic lavage. These changes were not observed in our control groups. Standard bowel preparation altered the diversity of mucosa-associated microbiota. Taxonomic classification did not reveal significant changes at the phylum level, but there were differences observed at the genus level.Standard bowel cleansing preparation altered the mucosal-adherent microbiota in all of our subjects, although the degree of change was variable. These findings underscore the importance of considering the confounding effects of bowel preparation when designing experiments exploring the gut microbiota. The ability to study the enteric microbiota in health and disease has rapidly evolved, largely through the development of non-cultivation based molecular approaches that provide information on the composition and structure of complex microbial communities. In this regard, numerous studies have surveyed the colonic microbiota to determine its relationship to the host and how perturbations of it affect or are affected by diseases such as antibiotic associated colitis, inflammatory bowel disease, pouchitis, and obesity Mai and colleagues It is now recognized that the gut microbiota is a unique ecosystem consisting of numerous microbial populations working together to carry out important physiological functions. Microbial species present in the colon lumen and adherent to the colonic mucosa are most often considered for studies of the human gut microbiota Twelve healthy individuals between the ages of 25–48 years were recruited at the University of Chicago Medical Center for this study. Written consent was obtained from all subjects prior to sample collection. Subjects were excluded if they had received antibiotics in the 6 months prior to the study. The Institutional Review Board of the University of Chicago Medical Center approved this study protocol (IRB#: 15006A).The study design is summarized in In order to better understand changes in the microbiota observed during the first phase of our study, we designed a second phase. The second phase was designed to control for external factors (such as time and diet) which might alter the microbiota. In the second phase, seven healthy individuals were randomly assigned into three groups. All subjects underwent an un-prepped flexible sigmoidoscopy with mucosal biopsies (pre-prep). In order to minimize any potential variation of enteric microbial communities over time, a second flexible sigmoidoscopy was performed in all subjects within 1 week. In the first sub-group, three subjects were given a 24-hour clear liquid diet and a standard polyethylene glycol-based bowel cleansing preparation prior to the second sigmoidoscopy (post-prep). In the second sub-group, two subjects underwent a second un-prepped flexible sigmoidoscopy and were instructed to make no changes to their diet prior to the second sigmoidoscopy. In the third group another two subjects underwent a second un-prepped flexible sigmoidoscopy and were instructed to follow a clear liquid diet for 24 hours prior to the second sigmoidoscopy. All biopsies were obtained 20 cm from the anal verge.Mucosal biopsy samples were homogenized in 1 ml extraction buffer  containing 20 ul proteinase K (20 mg/ml). A slurry of 500 ul of 0.1-mm-diameter zirconia/silica beads  were added to the extraction tubes. Tubes were placed on a Mini-Beadbeater-8 cell disrupter (BioSpec Products) for 5 minutes lysing the microbial cells. After overnight incubation at 55°C, extraction with phenol: chloroform: isoamyl alcohol, and precipitation with ethanol were performed. Isolated DNA was dissolved in TE buffer and stored at −80°C 5′-AGAGTTTGATCCTGGCTCAG-3′) labeled with 6′ carboxyfluorescein (6-FAM) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) for the bacterial domain MspI, RsaI, or HhaI (New England Biolabs Inc.) and subsequently subjected to capillary electrophoresis using the Applied Biosystems DNA sequencer 3130.16S rRNA gene sequences were amplified from DNA templates using broad-range primers 8F  profiles were constructed for samples taken from the 5 patients enrolled in the initial phase of the study. Restriction-digestion fragment presence and abundance was determined using GeneMapper software (Applied Biosystems). Raw electropherograms were analyzed for artifacts, such as electrical anomalies, optical cross-talk between the capillaries, baseline drift, fluorescence of non-FAM-labeled contaminants, and distortions of the sizing ladder. Terminal restriction fragment (TRF) data generated by GeneMapper were filtered and binned by the method developed by Abdo et al Library cloning and sequencing were performed on all samples obtained from the 7 subjects enrolled in the second phase of the study. Unlabeled PCR primers 8F and 1492R were used to amplify 16S rRNA gene sequences from the samples using the same protocol followed for T-RFLP analysis. PCR products were purified with the QIAquick gel extraction kit  and cloned into pCR-2.1-TOPO® using the TOPO-TA Cloning Kit  according to the manufacturer's instructions. From each library, 288 colonies were picked randomly and the inserts sequenced using 8F. DNA sequencing was performed at the University of Chicago's Cancer Research Center DNA Sequencing Facility using the Applied Biosystems 3730XL DNA Analyzer.http://rdp.cme.msu.edu/pipeline) by base-calling, quality-trimming and alignment. Raw sequence data were processed and trimmed according to quality scores through an automated workflow (RDP Pipeline Tool via myRDP) available from the Ribosomal Database Project (RDP) II website (http://rdp.cme.msu.edu/) The 16S rRNA gene sequences were analyzed as previously described MspI and RsaI. There was no clustering in the profiles where HhaI was used. MspI digestion provided the clearest clustering of individual's microbial communities and these profiles were used for further analyses.In the first phase of the study, there was clustering of each individual's microbial communities in profiles where the 16S rRNA gene was digested with A dendrogram comparing the microbial communities in biopsies obtained from phase 1 subjects before and after bowel preparation is shown in In the second phase of the study, 16S rRNA gene clone library and sequencing analysis of the 14 samples obtained from the 7 subjects enrolled in the experimental and control groups. A total of 4,032 clones were randomly picked from 14 clone libraries and sequenced. After quality control and sequence assembly, 3158 clones yielded approximately 650 base pairs of partial 16S rRNA gene sequences that were used for analysis . 16S rRNBecause we observed a reduction in diversity after bowel preparation in all three subjects in our experimental group, we used a one-tailed t-test to measure this effect. The Shannon diversity index was significantly lower in the samples collected after bowel preparation compared to those collected before preparation (p = 0.04). There was not a significant decrease in the Shannon diversity index in samples collected during the second flexible sigmoidoscopy compared to those collected during the first flexible sigmoidoscopy in either control group  was used to assess and compare the total diversity of mucosa-associated microbiota between samples. As shown in Because the richness analysis does not account for the similarity of phylogenetic composition between samples, we used Unifrac based clustering analysis to define the compositional difference between samples. A dendrogram was constructed from the sequencing data and showed that the microbiota from each individual consistently clustered together. This clustering of the microbiota in individuals was not seen in samples subjected to T-RFLP analysis, . We beliBlautia was decreased by bowel preparation in subjects 1 and 2, but not changed in subject 3. Butyricicoccus was decreased in subjects 1 and 3, but not detected in subject 2. However, Mucispirillum, a group of bacteria present in low abundance in the un-prepped colon, consistently disappeared completely after bowel preparation in all three subjects.The taxonomic outlines of each sample were also examined. The majority of organisms in all samples were classified into two phyla Firmicutes and Bacteroidetes, which is consistent with other studies using biopsies from prepped human colon. Organisms from the phyla Proteobacteria, Verrucomicrobia, Fusobacteria, Actinobacteria, Acidobacteria, and Deferribacteres were minor populations in these samples. As shown in Significant advances have been made in technology and bioinformatic analysis allowing investigators to study and better understand complex microbiota. Despite these advances, many challenges remain. Among these challenges, include obtaining undistorted and representative samples from the human gut. While many researchers have suspected that colonic lavage distorts the enteric microbiota, there are many challenges in obtaining samples from an un-prepped colon. Our study demonstrates that colonic lavage may distort both the diversity and structure of the enteric microbiota and these findings question the reliability of sampling by luminal fluid aspiration and mucosal biopsy after colonic lavage.We observed a reduction in overall diversity after colonic lavage in the mucosal samples obtained from three of the five subjects in the first phase of our study. Microbial diversity appeared to increase after colonic lavage in the other two subjects. This observed increase in diversity in the latter two subjects likely accounts for the similar Shannon diversity scores in the prepped and un-prepped samples. These initial observations prompted us to repeat our study and include control groups to determine if there are normal fluctuations in an individual's colonic microbiota and to determine if a clear liquid diet alone alters the enteric microbiota. In the second phase of our study, we shortened the time interval between flexible sigmoidoscopies to minimize any potential fluctuations in an individual's colonic microbiota over time. In order to achieve greater resolution than that achieved with T-RFLP analysis, 16S rRNA gene clone library and sequencing analysis were performed on samples collected from subjects enrolled in the second phase of the study. There was a decrease in richness and microbial structure similarity after colonic lavage. These changes were not observed in the two control groups, confirming that the trend towards a decrease in microbial diversity observed in patients taking a bowel prep is not due to clear liquid diet alone or normal fluctuations in the colonic microbiota.Although our sample size is small, it appears that colonic lavage does affect the enteric microbiota, albeit this effect is not consistent among individuals. The variation in response to colonic lavage observed in our study may be attributable to large differences between individual's enteric microbiota, effectiveness and completeness of colonic lavage, and inter-individual differences in factors that may influence the enteric microbiota such as diet, medications, and lifestyle.Previous studies of the human microbiota demonstrated a modest diversity of colonic microorganisms relative to other microbial communities, 62% of which appeared to be unique based on 16S rRNA gene sequence Our study demonstrates a variable treatment effect of colonic lavage on the mucosa-associated enteric microbiota independent of the clear liquid diet or normal variation of the enteric microbiota with time. Based on our findings, we believe that future studies of the enteric microbiota should take into consideration the confounding effects of colonic lavage. Studies that had previously acquired microbiota samples under these circumstances may have to be revisited under less perturbing conditions. We recommend that future investigations of the human enteric microbiota include un-prepped subjects in whom the natural state of the colonic microbiota can be preserved and observed. Acquisition of left sided colon samples should not be a major problem, but obtaining right side colonic samples will be more technically demanding; potentially requiring conscious sedation and increasing risk and duration of colonoscopic procedures. Nonetheless, in the hands of an experienced endoscopist, full colonoscopy in un-prepped individuals is feasible. Our group has an 80% success rate reaching the cecum in un-prepped patients.In summary, we report that the routine practice of colonic lavage may significantly alter the mucosa-associated microbiota of the distal human colon. While the effects are obvious in some individuals, the effects of colonic lavage can be unpredictable. Given that colonic lavage has the potential to distort the enteric microbiota, we recommend that future studies of the human enteric microbiota be performed on the un-prepped colon where the natural state of both luminal and mucosa-associated microbiota is most likely to be retained.Figure S1Relative abundance of bacterial genus in all samples. (Sub represents subject. FS1 represents the first flexible sigmoidoscopy and FS2 represents the second flexible sigmoidoscopy.)(TIF)Click here for additional data file.Figure S2Effect of polyethylene glycol prep on murine colon. Following purge with polyethylene glycol prep, mice had dramatic loss of mucosal-associated microbes and destruction of biofilm. Depletion of goblet cells was also noted in post-prep sample. These changes were not seen in the mice lavaged with normal saline.(JPG)Click here for additional data file.Table S1(1FS represents Subgroup 1 in Phase II. 2FS represents Subgroup 2 in Phase II. 3FS represents Subgroup 3 in Phase II. Sub is abbreviation for Subject.)(DOC)Click here for additional data file.


Streptomyces species is co-ordinated with differentiation events. An understanding of the genetic circuits that regulate these coupled biological phenomena is essential to discover and engineer the pharmacologically important natural products made by these species. The availability of genomic tools and access to a large warehouse of transcriptome data for the model organism, Streptomyces coelicolor, provides incentive to decipher the intricacies of the regulatory cascades and develop biologically meaningful hypotheses.The onset of antibiotics production in Streptomyces coelicolor probed across multiple stress and medium conditions, were investigated. Information based on transcript and functional similarity was used to update a previously-predicted whole-genome operon map and further applied to predict transcriptional networks constituting modules enriched in diverse functions such as secondary metabolism, and sigma factor. The predicted network displays a scale-free architecture with a small-world property observed in many biological networks. The networks were further investigated to identify functionally-relevant modules that exhibit functional coherence and a consensus motif in the promoter elements indicative of DNA-binding elements.In this study, more than 500 samples of genome-wide temporal transcriptome data, comprising wild-type and more than 25 regulatory gene mutants of Streptomyces coelicolor. The functionally-relevant modules identified in this study pose as potential targets for further studies and verification.Despite the enormous experimental as well as computational challenges, a systems approach for integrating diverse genome-scale datasets to elucidate complex regulatory networks is beginning to emerge. We present an integrated analysis of transcriptome data and genomic features to refine a whole-genome operon map and to construct regulatory networks at the cistron level in  Streptomyces coelicolor, the model organism for this high G+C genus, contains 7825 genes. The genome contains more than 20 secondary metabolite clusters and 965 genes encoding proteins predicted to have a regulatory role C[AG][N16-17]CGTA CGTA  is similression) , known tp-value threshold of 1.0 × 10-4, 146 network modules were enriched in 33 different protein classes. Twenty-five network modules were enriched in the secondary metabolism protein class. Additionally, 16 network modules were enriched in the polyketide synthase protein class. For the classification using GO terms, at a p-value threshold of 1.0 × 10-4, 115 network modules were enriched in 67 GO terms. The term that appeared as enriched in the most number of network modules (13) was NADH dehydrogenase (ubiquinone) activity. The complete list of 188 unique enriched network modules can be found in additional file A functional module is a group of components and their interactions that can be attributed a specific biological function . We inveFunctionally coherent network modules that also contain a consensus sequence are highly probable to indicate true interactions. Thus, in the above functionally coherent modules, those containing a consensus sequence were identified. A total of 20 network modules contain a consensus sequence in the upstream region of all of its members and present biological enrichment  retained the same prediction. Of the 526 differences, 422 correspond to adjacent genes predicted to be co-transcribed in the current prediction, but not in the previous one. Only 104 differences corresponded to adjacent genes not predicted as co-transcribed in the current work and predicted as co-transcribed in the previous work. Most of these pairs had a low expression correlation in the expanded dataset.The training set for the current predictions consisted of 425 KOPs and 131 NOPs. The KOPs consist of literature-reported operons as well as those experimentally verified in our previous study [k-means, or self-organizing maps) where cistrons or genes are assigned to mutually exclusive groups, a cistron can participate in multiple network modules, thus linking them and allowing a cistron to engage in different biological functions. ARACNE identified a Streptomyces coelicolor transcriptional network with scale-free connectivity distribution. Scale-free architecture has been noted in other networks derived from transcriptional interactions [An advantage of the algorithm employed in this study (ARACNE) is that unlike clustering algorithms .Click here for file


Somatoform dissociation is a specific form of dissociation with somatic manifestations represented in the form of ‘pseudoneurological’ symptoms due to disturbances or alterations of normal integrated functions of consciousness, memory or identity mainly related to trauma and other psychological stressors. With respect to the distinction between psychological and somatoform manifestations of dissociation current data suggest a hypothesis to which extent mild manifestations of ‘pseudoneurological’ symptoms in healthy young population may be linked to stress-related psychopathological symptoms or whether these symptoms more likely could be attributed to unexplained somatic factors.With this aim we have assessed the relationship between somatoform dissociation and stress-related psychopathology  in a group of 250 healthy non-psychiatric and non-clinical young adults.Results of this study show that the symptoms of somatoform dissociation are significantly linked to stress-related psychopathology.Findings of this study show that the ‘pseudoneurological’ symptoms may be linked to stress-related psychopathological processes which indicate that also mild levels of stress may influence somatic feelings and may lead to various somatoform dissociative symptoms. Somatoform dissociation has been proposed as a concept describing specific forms of dissociative symptoms experienced as somatic disturbances due to alterations of normal integrative functions of consciousness, memory or identity related to stressful experiences -4. FrequAlthough the concept of somatoform dissociation seems to be clinically relevant, the distinction between psychological and somatic forms of dissociation represents a fundamental problem whether dissociative symptoms, reflecting disorders of conscious awareness, are always “psychological” in nature or they may have somatic manifestations mediated by somatization or conversion mechanisms -8. With With the aim to test the hypothesis we have assessed the relationship between ‘pseudoneurological’ symptoms represented by somatoform dissociation questionnaire, and stress-related psychopathological symptoms  in a group of 250 non-psychiatric and non-clinical healthy young adults, who represent population particularly vulnerable to stress influences.Participants of this study were selected within the framework of European Longitudinal Study of Parenthood and Childhood (ELSPAC). The longitudinal study started in 1992 and included few European countries, and was organized in the UK and Czech Republic. Cohort of the study was selected randomly in the population of the city of Brno in the Czech Republic based on voluntary agreement provided by parents awaiting a newborn child. In the present study, as a part of ELSPAC, data of 250 non-psychiatric and non-clinical healthy young adults were collected. Based on anamnestic data exclusion criteria in this study were presence of psychiatric, neurological, internal and other somatic disorders. The participants were 101 men and 149 women . Data characterizing participant’s somatoform dissociation and other psychopathological manifestations related to symptoms of traumatic stress, depression, anxiety and alexithymia were acquired. All the participants gave written informed consent and the study was approved by Masaryk university ethical committee. All the data used in the study were acquired at the Institute of Preventive and Social Pediatrics, Faculty of Medicine, Masaryk University, Brno and data acquisition and processing were done by mental health professionals at the Institute.Somatoform dissociative symptoms were assessed using the 20-item self-reported somatoform dissociation questionnaire SDQ-20 ,16,17. SFor investigation of childhood traumas, TSC-40 (Trauma Symptom Checklist) -18 was uFor the assessment of depressive symptoms Czech version of Beck depression inventory BDI-II ,17,19 waLevels of anxiety symptoms were assessed using the Czech version of the Zung Self-Rating Anxiety Scale (SAS)  ,21. The Alexithymia was assessed using the Czech version of the 20-item Toronto Alexithymia Scale (TAS-20)  ,22. EachThe Czech versions of the all questionnaires were originally created in 2005 by translation from the English original and then back-translated into English and the resulting document was compared with the original by a native English speaker. The Czech version were then tested on a sample of 400 persons selected from general population and on samples of psychiatric and neurological patients, and results using these tests were published ,17,21.Statistical evaluation for the results of SDQ-20 and other psychometric measures included descriptive statistics, Mann-Whitney test for independent samples, Kruskal-Wallis ANOVA, Spearman correlation coefficients and multiple linear regression analysis. The non-parametric analyses were preferred because SDQ-20 data have not normal distribution. All the methods of statistical evaluation were performed using the software package Statistica version 6. To prevent Type II error which would disable to reject null hypothesis that symptoms of somatoform dissociation are not linked to stress-related psychopathological symptoms we performed Power Analysis and assessed the effect sizes characterizing differences between means of the subsamples with higher and lower levels of somatoform dissociative symptoms.To test psychopathological symptoms with respect to occurrence of pseudoneurological symptoms related to somatoform dissociation we have used descriptive statistics for the whole sample , BDI-II , SAS  and TAS-20 . These correlations show that SDQ-20 pseudoneurological symptoms exhibit significant and proportional relationship to symptoms of traumatic stress, depression, anxiety and alexithymia. In the analysis we have not found specific relationships between SDQ-20 and the subscales of the TSC-40 and TAS-20. All correlations between SDQ-20 and the subscales were statistically significant but did not show statistically significant differences between the correlation coefficients.st from 20 to 25; 2nd from 25 to 30; 3rd from 30 to 35; and 4th including all SDQ-20 scores from 36. To analyze differences in TSC-40, BDI-II, TAS-20 and SAS between the subgroups defined according to criteria of SDQ-20 subgroups we have used Kruskal-Wallis ANOVA. The results show that increased SDQ-20 scores in these subgroups are significantly linked to increased values of psychopathological symptoms measured by TSC-40, BDI-II, TAS-20 and SAS  which indicate that increased values of somatoform symptoms are related to continuously increased psychopathological symptoms.To confirm and further analyze results obtained by correlation analysis we have divided the whole sample according to SDQ-20 score with respect to interquartile range Table  into 4 sf .To analyze effects of TSC-40, BDI-II, TAS-20 and SAS on SDQ-20, we have used a multiple linear regression that may be useful to know whether stress-related psychopathological symptoms in their specific interactions are proportionally linked to increased levels of SDQ-20. The result shows that multiple R = 0.60 is statistically significant  which enables to define SDQ-20 as a linear function of four variables SDQ-20 = Results of this study show that the ‘pseudoneurological’ symptoms described in the context of somatoform dissociation are significantly and proportionally associated with stress-related psychopathology and that also relatively mild stressors may be linked to somatic manifestations. A limitation of this study may be occurrence of “medically unexplained symptoms”  that mayNevertheless with respect to the hypothesis findings of this study show that the “pseudoneurological” symptoms likely at least in part have psychological origin and can be explained by dissociative mechanisms in their somatic form within the framework of somatoform dissociation. Although some somatic factors influencing the symptoms cannot be rejected, predominant influence on these pseudoneurological symptoms likely may be linked to stress. Important aspect of this association presents the relationship between the pseudoneurological symptoms and alexithymia suggesting that loss of inner ability to distinguish, experience and interpret internal emotional states and feelings typical for alexithymia  is linkeIn addition, dissociated mental states have sensory, emotional and cognitive elements that may be misinterpreted and experienced as physical, and due to this misinterpretation physical and emotional experience can become confused and one of them may turn into the other . For exaThis influence of stress and dissociation on somatic experience and bodily functions is in agreement with growing evidence that for example orbital prefrontal areas regulate affect, motivation, and bodily state and that early relational traumatic stress is specifically imprinted into the right brain, which is dominant for autobiographical memories . Those aResults of this study indicate that also mild levels of stress in the mind of healthy people may be linked to somatic pseudoneurological symptoms and that somatoform dissociation may present an important mediating factor which may explain relationship between mental stress and somatic symptoms. Results of the present study together with other reported findings strongly suggest that clear diagnostic distinction based on available evidence about somatoform dissociation presents an important issue for clinical practice. Mainly these research findings could be useful for description and classification of diseases that in their current forms  only partially take into account influences of mental states on somatic functions and symptoms. A detailed analysis which somatic symptoms might be with high probability attributed to somatoform dissociation likely would have high clinical impact for differential diagnostics and could provide useful diagnostic instrument.The authors declare that they have no competing interests.PB, LK, JR- study design and data analysis; PS- collecting data; PB- wrote the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/13/149/prepub


We thus found SecA to be both necessary and sufficient for translocation of secretory proteins with hydrophobic signal sequences, whereas SRP and its receptor improved translocation efficiency. This SRP-mediated boost of translocation is likely due to the early capture of the hydrophobic signal sequence by SRP as revealed by site-specific photo cross-linking of ribosome nascent chain complexes.The Sec translocon of bacterial plasma membranes mediates the linear translocation of secretory proteins as well as the lateral integration of membrane proteins. Integration of many membrane proteins occurs co-translationally via the signal recognition particle (SRP)-dependent targeting of ribosome-associated nascent chains to the Sec translocon. In contrast, translocation of classical secretory proteins across the Sec translocon is a post-translational event requiring no SRP but the motor protein SecA. Secretory proteins were, however, reported to utilize SRP in addition to SecA, if the hydrophobicity of their signal sequences exceeds a certain threshold value. Here we have analyzed transport of this subgroup of secretory proteins across the Sec translocon employing an entirely defined  The heterotrimeric SecYEG translocon located in the inner membrane of Gram-negative bacteria is used by two different groups of proteins to be exported from the cytoplasm of these organisms: secretory proteins destined for the periplasmic space or the outer membrane are translocated through the pore of the Sec translocon, whereas inner membrane proteins exit laterally from the Sec translocon into the lipid bilayer. Both classes of proteins carry characteristic targeting signals. In the case of secretory proteins these are classical N-terminal signal sequences consisting of a positively charged N-region, a hydrophobic core, and a polar C-region. These signal sequences are typically cleaved off after transport. Membrane proteins instead are recognized via non-cleaved hydrophobic transmembrane helices called signal anchor sequences. For a recent comprehensive review on the structure and function of the Sec translocon, see ref. E. coli. Binding of SRP occurs co-translationally at the ribosome. The resulting complexes between SRP and ribosome-nascent chains (RNCs) are thought to dock at the membrane-bound SRP receptor (SR), called FtsY in bacteria, via a direct interaction between the Ffh and FtsY proteins The different targeting signals engage two diverse targeting routes. Signal anchor sequences of membrane proteins, which in general are more hydrophobic than classical signal sequences, recruit the signal recognition particle (SRP) consisting of the Ffh protein and the 4.5S RNA in On the contrary, the comparably less hydrophobic signal sequences of secretory proteins are not stably bound by SRP at the ribosome and hence elicit a post-translational targeting mode. This usually involves protection by chaperones such as SecB and Trigger factor until the signal sequence binds to the motor protein SecA that has a high affinity for the Sec translocon.SecA was, however, found to be required also for the assembly of SRP-dependent inner membrane proteins if these harbor extended hydrophilic loops that need to cross the membrane during assembly in vitro system from entirely defined components. This system was based on the PURE (Protein synthesis Using Recombinant Elements) translation system In order to be able to discriminate between SRP- and SecA-dependent functions during targeting and translocation of secretory proteins with highly hydrophobic signal sequences we set up an E. coli proteins SfmC and TorT E. coliE. coli  into ribosome-associated nascent chains (RNCs) of SfmC via the suppression of amber stop codons engineered into the sfmC gene. To this end, a pBpa-accepting suppressor tRNA encoded by plasmid pEVOL-pBpF E. coli cells that had been transformed with pEVOL-pBpF. The same plasmid also encodes a pBpa-specific aminoacyl-tRNA synthetase, which was modified to contain a C-terminal His-tag to allow its purification and use in the PURE system. SfmC-RNCs could quantitatively be synthesized in an oligodeoxynucleotide-dependent manner We surmised that the beneficiary effect by SRP/SR on the translocation of SfmC might be mediated by the recruitment of Ffh to the hydrophobic signal sequence at the ribosome. In order to demonstrate this directly, we expanded the PURE system such as to allow the site-specific incorporation of the photo-activatable cross-linker arrow). Upon supplementing the defined protein set of the PURE system with purified Ffh and FtsY, a prominent cross-linking product of the SfmC-RNCs appeared (asterisk). Its size of about 60 kDa was indicative of an adduct between the SfmC-RNCs (about 13 kDa) and Ffh (48 kDa) and as such it was recognized by anti-Ffh antibodies (lane 11) and did not depend on the presence of FtsY (not shown). The Ffh cross-link markedly decreased in intensity upon addition of SecYEG proteoliposomes, (compare lanes 5 to 6) and even disappeared almost completely when purified SecA was added together with Ffh, FtsY, and proteoliposomes (compare lanes 3 to 4). Under these conditions, a new high molecular mass adduct appeared  that was recognized by anti-SecA antibodies (lane 14). Contacts to SecA were, however, only obtained in the presence of SecYEG proteoliposomes  implying that only membrane-associated SecA interacted with the signal sequence of the SfmC-RNCs in these conditions. The fact that the interaction with SecA was paralleled by a drastic decrease in Ffh contacts would be consistent with an SRP-dependent targeting to membrane-bound SecA. Nevertheless Ffh and FtsY were dispensable for the SecA contacts to form  in accordance with the finding depicted in To identify binding partners of the SfmC signal sequence before and after membrane targeting, we synthesized SfmC-RNCs of 126 amino acid length that carried pBpa in the hydrophobic core of their signal peptide , arrow. asterisk, lane 1) that was totally dependent on the addition of Ffh (cf. lane 5). Hence a stretch of SfmC extending from its signal sequence up to position V58 was exposed to Ffh after having emerged from the ribosome. The cross-linker in position V58 also yielded the prominent 100 kDa adduct if SecA and SecYEG proteoliposomes were provided . A slight interaction of SecA with V58 in the early mature part of SfmC was obtained even in the absence of SecYEG proteoliposomes, provided that the competing Ffh was missing (compare lanes 1 and 5). This finding is in line with the idea that SecA might interact with nascent chains due to its association with ribosomes (see Discussion). The shift of the cross-linking partners from Ffh to SecA observed upon addition of SecYEG proteoliposomes (compare lanes 1 and 2) would be expected if Ffh/FtsY was to target SfmC-RNCs to the SecYEG translocon. In fact, SfmC-RNCs with pBpa at position V58 also efficiently cross-linked to a resident protein of the SecYEG proteoliposomes (dot), which by immunoprecipitation was identified as SecY (lane 8).We then moved the cross-linker from the signal sequence downstream to position V58 in the early mature part of SfmC . Much liE. coliSignal sequences, whose hydrophobicity exceeds a certain threshold level, have been proposed to direct secretory proteins to the SRP-dependent co-translational export pathway of By use of an experimental system that allows studying the individual contributions of Ffh, FtsY and SecA to translocation across the SecYEG translocon, we were now able to establish the dominant role of SecA in the export of this kind of secretory proteins. Thus we could demonstrate that SecA is even sufficient for their translocation, while SRP and SR turned out to render transport more efficient. The latter finding would be consistent with the notion that because of inherently rapid folding kinetics, some secretory proteins require a hydrophobic signal sequence in order to co-translationally enter their export route and thereby escape premature folding in vivo analyses in vivo is present at about 10fold higher concentrations than Ffh in vitro analysis employing highly defined experimental conditions demonstrate that the SRP dependence of secretory proteins with highly hydrophobic signal sequences is a result of a co-translational recognition of the hydrophobic signal sequence but that it is no prerequisite for the translocation of those secretory proteins, which can be executed solely by SecA.Channeling secretory proteins into the SRP/SR-dependent co-translational pathway invokes their recognition early during translation at the ribosome. We therefore analyzed RNCs carrying a protein with a hydrophobic signal sequence (SfmC) and could show a definite interaction of its signal sequence with the Ffh protein, which is in clear contrast to our previous data obtained with the less hydrophobic signal sequence of OmpA E. coli strains DH5α HisEG all described in ref. in vitro transcription of plasmid pT7/T3α19 in vitro from plasmids pKSM717-OmpA sfmC and torT genes were amplified from plasmids pCA24N-SfmC and pCA24N-TorT The precursors of OmpA, SfmC and TorT were synthesized sfmC gene of pET-SfmC at positions encoding I12 and Y14 within the signal sequence  using the primers SfmCI12for, SfmCI12rev, SfmCY14for, SfmCY14rev .Preparation of SecYEG proteoliposomes was performed as described In vitro transcription and translation assays were performed in the PURE system as described previously GATAAAATCGCCAGTTGCAAAAC, 0.3 μg anti-10Sa RNA oligodeoxynucleotide TTAAGCTGCTAAAGCGTAGTTTTGGTCGTTTGCGACTA and 3 units RNaseH were additionally supplied in each 25 μl reaction according to For the synthesis of ribosome-associated nascent SfmC chains of 126 amino acid length, 4 μg of the oligodeoxynucleotide sup prepared as described For the site-specific incorporation of pBpa into amber stop codon variants of SfmC, 80 μM pBpa, 20 μg/mL pBpa-tRNA synthetase, and 40 μg/mL pBpa-tRNAProtein translocation was analyzed by proteinase K resistance