Silver thin films (thickness ∼200 nm) were primarily deposited on plasma cleaned glass substrates by the physical vapor deposition (PVD) technique. A pair of rectangular silver electrodes was formed by patterning silver film through standard optical lithography technique. An active layer of organic-inorganic composite for humidity sensing was later spun coated to cover the separation between the silver electrodes. The electrical characterization of the sensor was performed as a function of relative humidity levels and frequency of the AC input signal. The sensor showed reversible changes in its capacitance with variations in humidity level. The maximum sensitivity ∼31.6 pF/%RH at 100 Hz in capacitive mode of operation has been attained. The aim of this study was to increase the sensitivity of the previously reported humidity sensors using PEPC and NiPc, which has been successfully achieved.In this paper, we present the effect of varying humidity levels on the electrical parameters and the multi frequency response of the electrical parameters of an organic-inorganic composite (PEPC+NiPc+Cu Organic semiconductors are materials that can be deposited at low temperature through uncomplicated deposition methods like spin coating and drop casting. These features give organic semiconductors an economic and technological edge over conventional inorganic semiconductor sensing materials. Consequently, extensive R&D efforts have been directed towards various organic semiconductors and their composites to identify appropriate materials for humidity sensing applications –5. A gooIn the past few decades, conventional inorganic semiconductors, particularly porous silicon, have been extensively utilized for humidity sensing applications. The working principle of these sensors is the change in conductivity of the humidity sensing layer. However, problems associated with conventional humidity sensors are the considerably higher cost of pure base materials and the high temperature fabrication procedures needed –9. Organviz., capacitive, resistive, hydrometric, gravimetric, optical, and integrated types [Based on the operating principle, organic material based humidity sensors fall into various categories ed types –12. The ed types , roughlyed types . The pered types .2O), poly-N-epoxypropylcarbazole (PEPC) and mixtures of the latter two have already been reported as ambient sensitive organic semiconductors. The humidity sensing capability of a blend of Cu2O and PEPC has been investigated in our previous study and the bandwidth of the sensor was found to be 36-86% RH, whilst its sensitivity was found to be 0.83 pF/%RH [et al. [2O as humidity sensor, where Cu2O is used as template to enhance the surface roughness. PEPC and NiPc create charge-transfer complexes due to the partial transfer of electrons from PEPC into NiPc. Due to the formation of the PEPC-NiPc complexs the resistance of the composite also decreases, that allows the capacitance of the humidity sensor to increase. The purpose of this study is to tune the sensitivity, bandwidth and linearity in response characteristics of the humidity sensor, with the aim of increasing the sensitivity of the previously reported humidity sensors using PEPC and NiPc [Nickel phthalocynine (NiPc), copper oxide , whereas nickel phthalocyanine powder was purchased from Sigma Aldrich , and was used without further purification. PEPC however was synthesized in the laboratory, and its fabrication process has been described in detail elsewhere [2O-PEPC-NiPC, obtained using a Hitachi SU-1500 scanning electron microscope . The SEM micrographs show porosity in the structure, suggesting Cu2O-PEPC-NiPC thin film is ideal for humidity sensing studies. Thin films of organic-inorganic composite, Culsewhere . Figures2O, 4 weight% PEPC and 5 weight% NiPC were initially blended in Benzol . Unlike PEPC and NiPC, Cu2O is inorganic in nature and is not dissolved in the organic solvent Benzene. This results in a colloidal solution of Cu2O-PEPC-NiPC composite in Benzene. A thin film of this suspension served as a sensing layer in our fabricated humidity sensor. Commercially available glass slides, which were thoroughly cleaned ultrasonically with distilled water for 10 minutes and later dried in a dust free environment, were utilized as a substrate. Glass slides were further plasma cleaned for 5 minutes with the subsequent fabrication step. Thin films of silver (thickness ∼200 nm) were deposited on a glass substrate by a thermal evaporator. An Edward Auto 306 vacuum coater with a diffusion pumping system  was used, which also provided the facility of plasma cleaning of the substrate's surface. While depositing a silver thin film, the pressure inside the chamber was kept at 10−5 mbar, whereas the deposition rate was maintained at 0.1 nm/s.Seven weight% Cu3). The gap between the surface type silver electrodes was 20 μm, which was subsequently covered by solution of organic-inorganic composite Cu2O-PEPC-NiPC in Benzene. The active sensing layer of Cu2O-PEPC-NiPC with thickness of 3–5 μm (the results presented here are the average results for 4 μm thick film) and the surface area 4 mm × 4 mm, was obtained by the spin coating technique and was dried under ambient conditions. A schematic view of the fabricated Ag/Cu2O-PEPC-NiPC/Ag surface type sensor is shown in Silver thin films were later patterned by using standard optical lithographic techniques, thus producing a pair of silver electrodes on the glass substrate. The silver thin film was etched by wet etching, using liquid nitric acid  for the AC measurements of sensor was 1.0 V. The frequency response of the sensor was studied by the recording capacitance of the sensor over a wide range of operating frequency, i.e., 100 Hz, 1 kHz and 10 kHz. The characterization setup has been portrayed in The humidity sensor was subjected to electrical characterization in a sealed chamber with a controlled humidity environment. The chamber was fabricated in our device testing laboratory and it had inlet and outlet valves for gas flow. To create humidity in the chamber, nitrogen gas is passed through water and then injected into the chamber. Monitoring of temperature and humidity levels in the humidity chamber was done by a humidity meter . The capacitance of the sensor was measured by Escort ELC 3133A Bench LCR Meter . The root mean square value of the applied voltage . The capacitive humidity sensors, typically exhibit a non-linear response as a function of relative humidity. The reason of change in capacitance with RH may be attributed to the immense difference in the dielectric values of water and composite film. At 25 °C, dielectric constant of water is ∼80, however organic semiconductors generally have small dielectric values. With the increase in absorption of water molecules, water molecule layer gets stacked up on composite layer, changing the net dielectric permittivity of the film. Dielectric constant thus plays pivotal role in the variation of capacitance, as both are directly related to each other. The relation between dielectric constant and capacitance can be described by the following equation [dε, and wε are the dielectric permittivities of the composite film at dry and wet conditions, respectively. “n” is morphological factor.The changes in capacitance as a function of relative humidity, at three distinct AC frequencies of applied voltage equation :(1)CSC02O, and dipoles are formed in organic π-conjugated large molecules and polymeric semiconductor molecule, due to charge transfer complexes and dissociation of molecules due to the introduction of water contents in the composite film. Consequently it can be assumed that capacitance increases due to polarization in composite film and dipole formation  in water molecules.Variation in capacitance is also associated with the polarization of the composite film . PolarizAs it is well understood that organic semiconductors are unsuitable for high frequency electronics applications such as high frequency switching devices . Hence oThe response and recovery time is an important characteristic for evaluating the performance of humidity sensors. The response time can be defined as the time required for a sensor output to change from its previous state to 90% of its final settled value. The recovery time of a sensor is reported as the time taken from the output of sensor to fall to 10% of final settled value. Step introduction and removal of measured variable, which in our case was relative humidity, is vital for response/recovery measurement. As portrayed in One of the most important challenges with humidity sensors based on the principle of absorption is hysteresis. Hysteresis is defined as the percentage difference in final settling point of relative humidity when approached from above to when it is approached from below. This phenomenon exists due to the formation of clusters of absorbed water in the active thin film and it depends upon the geometry of the pores. The larger pore size reduces the response time but they also reduce the sensitivity so it is difficult to make fast and sensitive humidity sensor. In our study, the hysteresis property of humidity sensor was studied by measuring capacitance at 100 Hz AC signal. Experimental data  showed t4.2O-PEPC-NiPC/Ag, was fabricated in planar configuration and its sensing performances were investigated experimentally, which included measurement of the capacitance of the sensor as a function of different environmental humidity levels. The sensor has been found to have good parameter stability and adequate sensorial properties. In general an increase in capacitance was observed with increasing humidity from 40% RH to 100% RH. Furthermore, as the composite film has earlier been found to be pressure sensitive as well [2O-PEPC-NiPC/Ag surface type humidity sensor would also help in developing a multifunctional sensor. The hysteretic behavior of the sensor showed an acceptable value of hysteresis (∼12%). The response and recovery time for were found to be 13–15 seconds. The comparison given in In this study, an organic-inorganic composite-based humidity sensor Ag/Cu as well , the fab


However, recent findings demonstrate that Trk receptors can be transactivated independently of their neurotrophin ligands.Antidepressant drugs (ADs) have been shown to activate BDNF (brain-derived neurotrophic factor) receptor TrkB in the rodent brain but the mechanism underlying this phenomenon remains unclear. ADs act as monoamine reuptake inhibitors and after prolonged treatments regulate brain in vivo and in vitro by employing several transgenic mouse models, cultured neurons and TrkB-expressing cell lines.In this study we examined the role of BDNF, TrkB kinase activity and monoamine reuptake in the AD-induced TrkB activation F616A mutant and TrkB overexpressing mice, we demonstrate that ADs specifically activate both the maturely and immaturely glycosylated forms of TrkB receptors in the brain in a TrkB kinase dependent manner. However, the tricyclic AD imipramine readily induced the phosphorylation of TrkB receptors in conditional bdnf−/− knock-out mice , indicating that BDNF is not required for the TrkB activation. Moreover, using serotonin transporter (SERT) deficient mice and chemical lesions of monoaminergic neurons we show that neither a functional SERT nor monoamines are required for the TrkB phosphorylation response induced by the serotonin selective reuptake inhibitors fluoxetine or citalopram, or norepinephrine selective reuptake inhibitor reboxetine. However, neither ADs nor monoamine transmitters activated TrkB in cultured neurons or cell lines expressing TrkB receptors, arguing that ADs do not directly bind to TrkB.Using a chemical-genetic TrkBThe present findings suggest that ADs transactivate brain TrkB receptors independently of BDNF and monoamine reuptake blockade and emphasize the need of an intact tissue context for the ability of ADs to induce TrkB activity in brain. TrkB (tropomyosin-related kinase B) neurotrophin receptor transduces intracellular signaling events that are critical for neuronal differentiation, survival and plasticity throughout life Abnormal TrkB receptor signaling has been linked to a number of central nervous system (CNS) diseases such as mood and memory disorders and addiction in vitro and in vivo. We show that both mature and immature forms of TrkB can be specifically tyrosine phosphorylated by ADs, but neither the endogenous ligand BDNF, nor the serotonin transporter (SERT), the principal target of many ADs, is required for this effect. However, the observation that ADs or serotonin (5-HT) or norepinephrine (NE) do not activate TrkB phosphorylation in vitro argues that ADs do not directly bind to TrkB receptors.In this study we have examined several potential molecular mechanisms of AD-induced TrkB activation in vivo. We have previously shown that several independent antibodies raised against phosphorylated tyrosines Y705/706 or Y816 within the intracellular domain of TrkB all show increased phospho-TrkB levels after acute and chronic AD treatment F616A knock-in mice F616A knock-in mice, NaPP1 treatment abolished this effect (flag-tagged TrkB receptors in adult neurons (TrkB.TK+) Previous studies suggest that BDNF-TrkB signaling is critical for the behavioral effects of ADs Figure S1A–B) and has been detected following TrkB immunoprecipitation and hybridization to pTyr antibodies Figure S1C). However, this band cannot be reliably detected by antibodies against the non-phosphorylated intracellular domain of Trk receptors . Nevertheless, acute fluoxetine treatment, with a dose and time point  which induced TrkB phosphorylation in mouse hippocampus Figure S2A). Furthermore, fluoxetine did not influence the activity of tissue plasminogen activator (tPA), the major regulator of pro-BDNF cleavage into mBDNF . These data suggest that ADs do acutely not influence BDNF levels or processing in brain.Previous studies have shown that acute AD treatment does not influence BDNF mRNA or protein levels 2L/2LCk-cre) lacking BDNF in forebrain regions in vivo. Imipramine readily induced tyrosine phosphorylation of both the mature and the immature glycosylated forms of TrkB in the hippocampus of conditional BDNF2L/2LCk-cre mice (bdnf+/− null mice and wild-type mice (data not shown). These data demonstrate that ADs activate TrkB receptors in the mouse brain in a manner independent of BDNF.Although BDNF is the main ligand of TrkB, recent evidence suggests that TrkB can also be activated independently of BDNF in neurons cre mice Figure 3via adenosine-2A signaling in the absence of BDNF in vitro and in vivo2A receptors with ZM241385 might block the acute effects of ADs on TrkB phosphorylation. We found that, imipramine increased the phosphorylation of TrkB receptors similarly in mice pretreated with saline or active dose Figure S3), suggesting that A2A receptors were not involved.Adenosine has been shown to transactivate TrkB receptors in vitroS4). We further tested whether ADs might potentiate the pTrkB response induced by a small dose of BDNF or whether depolarization of neurons might render them sensitive to ADs in vitro. Imipramine did not facilitate BDNF-induced TrkB phosphorylation in vitro (+), no significant changes in TrkB phosphorylation were seen , on TrkB receptor phosphorylation in SERT knockout mice (sert−/−). A 6–10 fold up-regulation of extracellular 5-HT levels in sert−/− mice sert−/− mice in a manner indistinguishable of the wild-type mice  and NE levels (by DSP4) and used selective 5-HT and NE transporter blockers citalopram and reboxetine, respectively. As noted before pCPA and DSP4 treated mice, respectively  . Taken together, these data suggests us that ADs do not directly bind to TrkB, but, instead, emphasize the importance of developmental processes and intact tissue context in the ability of small molecule weight drugs such as ADs to activate TrkB autophosphorylation in vivo.While all the different ADs readily induce TrkB autophosphorylation in rodent brain trkBF616A knock-in 2L/2LCk-cresert−/− knock-out mice Animal experiments - Wild-type (C57/BL6), TrkB.TK+ mutant In vivo drug treatments - Mice received a single i.p. injection of tested chemical or saline and after indicated time the mice were killed with CO2 and brain area of interest dissected on a cooled plastic dish. Next the samples were lyzed in NP++ buffer  and 2 mM Na3VO4), incubated on ice (>30 min) and centrifugated . The supernatant was processed further as described below. The following chemicals were used: fluoxetine-HCl (Orion Pharma), citalopram-HBr , imipramine-HCl, moclobemide (kind gift from F Hoffmann-La Roche Ltd), clomipramine-HCl, amitriptyline-HCl, reboxetine (GSK). Reboxetine and moclobemide were first stock-dissolved in DMSO, citalopram in ethanol, others directly in saline. In order to inhibit TrkB kinase activity prior drug administration in TrkBF616A knock-in mice, mice were pre-treated with 25 µM of 1NaPP1  for 7 days (in drinking solution) and further co-injected i.p. (83 ng/g) with imipramine or vehicle. ZM241358 was injected i.p. to block adenosine A2A receptors 30 min prior imipramine treatment pCPA  injections as described previously 2 50 mM Tris-HCl (pH 7.6) to remove Triton X-100 and the caseinolysis was allowed to occur by incubating the gels at +37°C for 16–24 h in the same solution. Caseinolytic areas were shown as translucent areas when the gels were stained with Coomassie Brilliant Blue.Tissue plasminogen activator (tPA) SDS-PAGE zymography - For tPA activity assay, freshly dissected brain samples were homogenized into buffer consisting of 0.1 M Tris-HCl (pH 8.0), 2.5% Triton-X-100, 10 µM leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethanesulfonylfluoride (PMSF). Samples and controls (human recombinant tPA) were loaded under non-reducing conditions in SDS-PAGE containing±human plasminogen (Sigma-Aldrich) and pre-heated non-fat dry milk at low current (∼15–20 mA) over night (O/N) at cold bath. Next the gels were rinsed thoroughly with 2.5% Triton X-100 to remove SDS and allow proteins to renaturate. Next the gels were rinsed thoroughly with 10 mM CaClIn vitro experiments - For the primary neuronal cultures, hippocampi or cortex was dissected from E18 rat embryos and the tissue dissociated in a papain solution , 250 glucose, ad 50 ml PBS; 10 min, 37°C). Next the cells were triturated and suspended in a medium containing 9.8 ml of Ca2+/Mg2+ free HBBS, 1 mM sodium pyruvate, 10 mM HEPES and 10 µl DNAse I. The cells were plated onto poly-L-lysine coated 12–24 well culture plates at a cell density of 0.5×106 ml−1  or 1×106 ml−1 . Cells were maintained in neurobasal medium  for 14 to 15 days in vitro (DIV) before treatments. Parental MG87 and MG87-trkB fibroblasts 2, +37°C) and were stimulated under confluent conditions. The following chemicals were used for the experiments: BDNF (Peprotech), imipramine, amitriptyline, desipramine, chlorpromazine, phenelzine, clozapine, lithium . After the treatments, the medium was discarded and the cells lyzed in NP++ buffer and processed for western blot analysis or for phospho-Trk ELISA described below.Figure S4A). Whereas in Trk expressing cells the assay readily detects BDNF- or NGF-induced Trk phosphorylation, such induction is not detected in cells not expressing Trk receptors (data not shown). Moreover, BDNF-induced TrkB phosphorylation is lost if the cells are pretreated with Trk kinase inhibitor k252a (data not shown). Stimulation experiments were initiated by adding the drugs at different concentrations onto confluent cell line cultures or with primary neurons. After indicated incubation period at +37°C, the medium was removed and the cells were lyzed with cold NP++ buffer (50–100 µl). In a set of experiments, these steps were carried out by using Biomek FX workstation (Beckman Coulter) for the liquid handling and incubation. Following >30 min incubation on ice all material were transferred to pre-coated  and pre-blocked  white 96-well Optiplate™ (PerkinElmer) plates and 2–3% BSA/PBS-T (+2 mM Na3VO4) added ad 200 µl. The plates were incubated O/N at +4°C and thereafter the wells washed with PBS-T (4×300 µl) and anti-phosphotyrosine antibody added to the wells . Following sequential washes and HRP-coupled tertiary antibody incubations  200 µl of ECL substrate (Pierce) was added to the wells and luminescence measured after 5 min with Varioskan Flash (Thermo Fisher Scientific) plate reader.Phospho-Trk ELISA - An enzyme-linked immunosorbent assay (ELISA) method was developed to easily measure the level of phosphorylated Trk receptors from cultivated cells (triticum vulgaris (Amersham or EY Laboratories). A set of lectin precipitated samples were incubated with endoglycosidase-A (Endo-H) according to manufacturer's instructions (New England Biolabs). Proteins were separated in a SDS-PAGE under reducing conditions and blotted onto polyvinylidene difluoride  membrane. After blocking , the membranes were incubated with primary antibodies: anti-pY705/6  Representative blot showing the time-response  of fluoxetine-induced  phosphorylation of TrkB and ∼105 kDa protein  in mouse hippocampus. B) Representative blots showing antidepressant-induced phosphorylation of ∼105 kDa in mouse hippocampus C) Representative blots showing imipramine-induced phosphorylation of ∼105 kDa in mouse striatum, midbrain and whole brain homogenate. Abbreviations: FLX = fluoxetine, SAL = saline; CIT = citalopram; AMI = amitriptyline; CLO = clomipramine; MOC = moclobemide; REB = reboxetine; MW = molecular weight; TrkB.extr = antibody directed against the extracellular portion of TrkB receptors.(TIF)Click here for additional data file.Figure S2Acute fluoxetine treatment did not regulate BDNF protein levels or tPA (tissue plasminogen activator) activity.A) Representative blot showing mature-BDNF specific band in western blot from brain homogenates and pro- and mature-BDNF specific bands from respective control lanes as detected with polyclonal BDNF antibody . Acute fluoxetine did not regulate mature-BDNF levels in mouse hippocampus. n = 6/group. B) Representative zymography showing caseinolysis at the level of recombinant tPA. Acute fluoxetine did not regulate tPA activity levels in mouse hippocampus. n = 6/group. Data is presented as percentage of control ± standard error of mean (SEM).(TIF)Click here for additional data file.Figure S32A receptor signaling does not prevent antidepressant-induced TrkB activation.Blockade of adenosine-A Acute imipramine treatment  induces essentially similar changes on TrkB phosphorylation in vehicle and adenosine-A2A receptor antagonist  pre-treated mice. A representative blot in left showing imipramine-induced phosphorylation of TrkB and ∼105 kDa protein in mouse brain. Data is presented as percentage of control/saline ± standard error of mean (SEM). *<0.05; two-way ANOVA with Newmann-Keuls post hoc test.(TIF)Click here for additional data file.Figure S4Phospho-TrkB enzyme-linked immunosorbent assay (ELISA).A) Dose-response of BDNF  on TrkB phosphorylation in TrkB expressing fibroblasts cultivated in 48-well plates. n = 4/group. B) Whereas BDNF produces robust TrkB phosphorylation in TrkB expressing fibroblasts cultivated in 24-well plates, all the tested drugs at selected doses did not have any effect on TrkB phosphorylation (compounds incubated for 15 min). n = 3/group. Data is presented as percentage of control ± standard error of mean (SEM). Abbreviations: IMI = imipramine; PHE = phenelzine; FLX = fluoxetine; CLOZ = clozapine; Li = lithium chloride; CHLOR = chlorpromazine.(TIF)Click here for additional data file.


Idiopathic pulmonary fibrosis (IPF) is a progressive and medically refractory lung disease with a grim prognosis. Although the etiology of IPF remains perplexing, abnormal adaptive immune responses are evident in many afflicted patients. We hypothesized that perturbations of human leukocyte antigen (HLA) allele frequencies, which are often seen among patients with immunologic diseases, may also be present in IPF patients.CO) compared to the 184 disease subjects who lacked this allele .HLA alleles were determined in subpopulations of IPF and normal subjects using molecular typing methods. HLA-DRB1*15 was over-represented in a discovery cohort of 79 Caucasian IPF subjects who had lung transplantations at the University of Pittsburgh (36.7%) compared to normal reference populations. These findings were prospectively replicated in a validation cohort of 196 additional IPF subjects from four other U.S. medical centers that included both ambulatory patients and lung transplantation recipients. High-resolution typing was used to further define specific HLA-DRB1*15 alleles. DRB1*1501 prevalence in IPF subjects was similar among the 143 ambulatory patients and 132 transplant recipients . The aggregate prevalence of DRB1*1501 in IPF patients was significantly greater than among 285 healthy controls . IPF patients with DRB1*1501 (n = 91) tended to have decreased diffusing capacities for carbon monoxide (DLDRB1*1501 is more prevalent among IPF patients than normal subjects, and may be associated with greater impairment of gas exchange. These data are novel evidence that immunogenetic processes can play a role in the susceptibility to and/or manifestations of IPF. Findings here of a disease association at the HLA-DR locus have broad pathogenic implications, illustrate a specific chromosomal area for incremental, targeted genomic study, and may identify a distinct clinical phenotype among patients with this enigmatic, morbid lung disease. Idiopathic pulmonary fibrosis (IPF) is a chronic, morbid, fibroproliferative lung disease that manifests with progressive pulmonary restriction and gas exchange abnormalities Despite extensive investigation, the etiology of IPF remains obscure. Although the pulmonary fibrosis that characterizes this disease is often considered to be uniquely dissociated from inflammatory processes HLA allele frequencies are often aberrantly distributed among patients with immunologic disorders We hypothesized that HLA frequency perturbations may also occur in IPF. Given the apparent prominence of CD4 T-cell responses in IPF The initial (discovery) IPF cohort consisted of 79 consecutive patients with end-stage pulmonary disease who had molecular HLA allele determinations during evaluations for lung transplantations at the University of Pittsburgh (U. Pgh.), beginning in March 2006.The subsequent disease validation cohorts were composed of IPF subjects for whom molecular HLA typing results and/or tissue specimens for HLA typing were available from the National Institute of Health (NIH) (n = 35), University of Chicago Medical Center (n = 32), Inova Fairfax Hospital (n = 20), and Stanford University Medical Center (n = 14). Subjects from Inova and Stanford were recipients of lung transplantations for end-stage IPF at their respective medical centers. IPF subjects from the NIH and University of Chicago were ambulatory clinic patients. In addition, data were compiled for 19 subjects who had lung transplantations for end-stage IPF at the U. Pgh. subsequent to the initial (discovery) analysis , and 76 ambulatory U. Pgh. IPF Clinic patients.Diagnoses were prospectively established in the IPF subjects by expert, specialized pulmonary clinicians, blinded to these experimental studies, who analyzed all clinical information, including medical histories and physical exams, pulmonary function tests (PFTs), laboratory studies that included serologic tests for conventional autoimmune syndromes, rheumatologist evaluations, chest radiographs, and computerized tomography (CT) scans that were interpreted by radiologists blinded to other study results. All IPF study subjects fulfilled consensus diagnostic criteria Right-heart catheterizations were routinely performed during assessments for lung transplantations  by cardiologists who were blinded to these HLA characterizations or knowledge of this study.Controls consisted of normal Caucasian subjects who had prior molecular HLA typing in the course of other investigations at the U. Pgh. (n = 196) Analyses are restricted to Caucasian subjects because <5% of lung transplantation recipients in the discovery cohort were members of minority groups, and HLA allele frequencies can vary greatly among racial/ethnic subpopulations Subjects gave written informed consent for these studies that were approved by the respective Institutional Review Boards of all the participating medical centers .HLA characterizations of the initial U. Pgh. IPF and normal cohorts were performed using DNA isolated from leukocytes, in sequence specific oligonucleotide probe assays Allele-disease associations, as well as comparisons of other dichotomous variables, were established by Chi square. Logistic regression analyses were used to generate odds ratios and 95% confidence intervals. Two-group comparisons of continuous variables were made by two-sample t-test. Factorial ANOVA was used for three or more group comparisons of demographic and physiologic data, with post-hoc comparisons by Bonferroni/Dunn. Analyses were conducted with StatView v5.0.1 . Alpha (p) values <0.05 were considered significant. Data are depicted as means±SE.CO%p), an indicator of intrapulmonary gas exchange, were significantly greater in the ambulatory IPF patients (48±2%) than among those patients who had lung transplantations (33±1%) (p<0.0001).A total of 275 IPF subjects were studied here . The IPFThe initial compilation of HLA alleles for the discovery cohort of 79 U. Pgh. transplantation recipients showed the prevalence of DRB1*15  was greater among the IPF patients than in a normal control population Based on these initial findings, validation cohorts were prospectively compiled, consisting of HLA data from IPF subjects at the NIH and results of DRB1*15 determinations using blood or tissue specimens from IPF patients at U. Chicago, Inova, Stanford, the IPF outpatient clinic at U. Pgh., as well as an additional volunteer U. Pgh. healthy control cohort. Those IPF and control specimens positive for DRB1*15 were further analyzed to characterize the specific alleles at this locus.DRB1*15 was present in 64 normal subjects (23%), and in 95 IPF patients (35%) (p = 0.0015). The DRB1*1501 allele accounts for most DRB1*15 expression in normal Caucasians The prevalence of DRB1*1501 within the aggregate IPF replication cohort (32.1%) was comparable to that of the original, discovery subjects (35.4%) (p = 0.60). Likewise, there was no significant difference of DRB1*1501 prevalence between IPF ambulatory patients (31.5%) and IPF transplant recipients (34.8%) (p = 0.55). The relative distributions of DRB1*1501 genotype frequencies among the respective control and IPF subpopulations were also similar in both discovery and replication cohorts . HoweverFive IPF patients were known to have first-degree relatives who died with lung disease(s) that could possibly have been IPF. Two of these patients had DRB1*1501. Inasmuch as was known, the remaining IPF cases were due to sporadic disease Four (4) pulmonary transplantation recipients with pre-operative diagnoses of IPF were excluded from analyses on the basis of lung explant histological evaluation that instead showed single cases each of: pulmonary vasculitic changes suggestive of a clinically unsuspected autoimmune disorder; predominant lymphangitic non-necrotizing granulomas and lymphocytic infiltrates more consistent with hypersensitivity pneumonitis or sarcoidosis; extensive necrotizing granulmatous bronchitis that was highly suggestive of an infectious etiology; and a clinically unsuspected, histoplasma pneumonitis. One of these excluded subjects (25%) had DRB1*1501.+ and DRB1*1501null subjects, respectively. Similarly, FVC%p did not consistently differ between the IPF patients with and without DRB1*1501 (There were no apparent associations between the presence (n = 91) or absence (n = 184) of DRB1*1501 among the IPF patients and either age (66±1 vs. 67±1 years) or gender , for the DRB1*1501RB1*1501 .CO%p values were consistently decreased among the DRB1*1501+ IPF subjects at each study site  and cumulative smoke exposures . DLCO abnormalities can also be attributable to secondary effects of pulmonary artery hemodynamics However, DLudy site . The meaThese findings show the HLA Class II allele DRB1*1501 is over-represented among Caucasian IPF subjects with highly variable disease severities at multiple U.S. medical centers . The preTwo distinct mechanisms may account for the frequently observed associations between unique HLA polymorphisms and various disease syndromes Alternatively, over-representation of a specific HLA molecule(s) in a disease cohort may be essentially unrelated to the unique antigen presentations of that HLA per se, but is instead a genetic “marker” denoting the presence of a pathogenic immunomodulatory gene(s) that is(are) in strong linkage disequilibrium (LD) with that HLA allele The human major histocompatibility (MHC) complex on chromosome 6p21.31 is characterized by the presence of numerous, extraordinarily polymorphic HLA alleles, and many other proximate immunoregulatory genes that are often in very strong LD Almost all previous HLA characterizations of IPF patients date from before the development of precise and definitive molecular methodologies that distinguish these alleles To our knowledge, contemporary analogous analyses using molecular techniques and current IPF case definitions are limited to a single cohort study of Mexican patients that reported various HLA alleles, including DRB1*01, DRB1*04, and DRB1*14, were over-represented in IPF The present study has several unique aspects. To our knowledge, the number of subjects with this uncommon disease that were analyzed here is unprecedented among analogous investigations . The finCO tended to be comparatively less among those IPF patients with DRB1*1501  function +CD28null T-cells, the daughter progeny of repetitive antigen-driven lymphocyte proliferations, and a frequent and specific finding of chronic immunologic diseases Although IPF is not widely considered to be an immunologic disorder, recent studies of patient-derived clinical specimens reveal the presence of abnormal adaptive immune responses among those afflicted by this disease. The majority of IPF patients have IgG autoantibodies against various autoantigens that are typically distinct from those described in classical autoimmune syndromes  Considerable efforts have been directed towards discovery of the genetic determinants for IPF


Idiopathic pulmonary fibrosis (IPF) is a severe, rapidly progressive diffuse lung disease. Several pathogenetic mechanisms have been hypothesized on the basis of the fibrotic lung damage occurring in this disease, and a potential profibrotic role of activated alveolar macrophages and their mediators in the pathogenesis of IPF was recently documented. This paper focuses on recent literature on potential biomarkers of IPF derived from activated alveolar macrophages. Biomarker discovery and clinical application are a recent topic of interest in the field of interstitial lung diseases (ILDs). Cytokines, CC-chemokines, and other macrophage-produced mediators are the most promising prognostic biomarkers. Many molecules have been proposed in the literature as potential biomarker of IPF; however, a rigorous validation is needed to confirm their clinical utility. Interstitial lung diseases (ILDs) are a heterogeneous group of rare diseases with different etiopathogenesis and clinical evolution . They inSeveral pathogenetic mechanisms have been postulated on the basis of fibrotic lung damage occurring in patients with IPF. The inflammatory theory has been partially replaced by the concept of aberrant wound healing due to interactions between epithelial cells and fibroblasts, determining uncontrolled chronic fibroproliferation . More reAnalysis of recent literature on biomarkers derived from activated alveolar macrophages in IPF is the main topic of this paper.In the last few years, molecules of different origin have been proposed as potential biomarkers of IPF. Fibrocytes, neutrophils, and alveolar epithelial cells are sources of mediators involved in the pathogenesis of IPF and proposed as candidate biomarkers, for example, KL6, surfactant proteins A and D, or circulating fibrocytes –26. Althhas been widely studied in BAL, serum, and tissue of IPF patients, proving a very promising biomarker derived from alternatively activated alveolar macrophages (Th2 cytokines and IL10 induce its biosynthesis) and highly expressed in the lungs , 33–35 . In 2006CC chemokine ligand 2  that together with CCL3 and CCL4, proved higher in BAL of IPF patients than controls [Alveolar macrophages can release other CC chemokines that could be implicated in fibrogenesis, including controls , 36–38. controls , 36. Shicontrols . Concentcontrols . The potcontrols . is a major mediator of inflammatory response. Produced by alveolar macrophages, epithelial cells, and other cell types, it functions as a chemoattractant for neutrophilic granulocytes, macrophages, endothelial cells, and mast cells and as a potent angiogenic factor . ConcentThe complex pathogenetic mechanisms and lack of diagnostic and prognostic biomarkers of IPF have driven proteomic research in the last few years. Several proteomic studies have been done, and a proteomic approach has been used to analyse protein composition of BAL from different ILD, including IPF, identifying proteins of interest , 45–49. Is a S100 protein produced by monocyte-macrophages, neutrophils, and other cells mainly in response to chronic inflammatory processes . S100A9 Another interesting protein identified by a proteomic approach in BAL of patients with IPF and differently expressed in patients than controls is macrophage migration inhibitory factor, MIF. MIF is a pleiotropic proinflammatory cytokine produced by activated macrophages and potentially involved in IPF pathogenesis for its numerous functions including regulation of cell redox status, inhibition of apoptosis, induction of matrix metalloproteinases, and regulation of Th1/Th2 immune responses . Its conThe recent literature on IPF indicates great interest in developing diagnostic and prognostic biomarkers and in defining the potential role of activated macrophages in the pathogenesis of IPF. Potential IPF biomarkers derived from activated alveolar macrophages, currently being studied, include CCL18, CCL2, IL-8, and calgranulin B. The discovery and development of biomarkers has to be conducted in a rigorous manner; studies on potential biomarkers have to be performed on a large scale, and appropriate statistical analysis is necessary to validate a molecule as a real biological indicator , 63. In Macrophage-derived biomarkers of IPF are proteins overexpressed in serum, BAL samples, or lung tissue of patients with IPF compared with controls. An alternative approach to identify potential bioindicators of IPF has been suggested by Ren et al. who reported evidence of impaired transcription in IPF macrophages. The authors concluded that the detection of a decrease in a specific protein produced by macrophages would be an alternative method to identify potential bioindicators of this severe interstitial lung disease .Sophisticated methods applied to different biological fluids are revealing interesting new groups of molecules that could provide future biomarkers of IPF, useful for fine phenotyping of patients and for clinical management of the disease.


Idiopathic Pulmonary Fibrosis (IPF) is characterized by profound changes in the lung phenotype including excessive extracellular matrix deposition, myofibroblast foci, alveolar epithelial cell hyperplasia and extensive remodeling. The role of epigenetic changes in determining the lung phenotype in IPF is unknown. In this study we determine whether IPF lungs exhibit an altered global methylation profile.LINE-1) retrotransposon while lung cancer samples did, suggesting that the global hypomethylation observed in cancer was not typical of IPF.Immunoprecipitated methylated DNA from 12 IPF lungs, 10 lung adenocarcinomas and 10 normal histology lungs was hybridized to Agilent human CpG Islands Microarrays and data analysis was performed using BRB-Array Tools and DAVID Bioinformatics Resources software packages. Array results were validated using the EpiTYPER MassARRAY platform for 3 CpG islands. 625 CpG islands were differentially methylated between IPF and control lungs with an estimated False Discovery Rate less than 5%. The genes associated with the differentially methylated CpG islands are involved in regulation of apoptosis, morphogenesis and cellular biosynthetic processes. The expression of three genes  with hypomethylated promoters was increased in IPF lungs. Comparison of IPF methylation patterns to lung cancer or control samples, revealed that IPF lungs display an intermediate methylation profile, partly similar to lung cancer and partly similar to control with 402 differentially methylated CpG islands overlapping between IPF and cancer. Despite their similarity to cancer, IPF lungs did not exhibit hypomethylation of long interspersed nuclear element 1 (Our results provide evidence that epigenetic changes in IPF are widespread and potentially important. The partial similarity to cancer may signify similar pathogenetic mechanisms while the differences constitute IPF or cancer specific changes. Elucidating the role of these specific changes will potentially allow better understanding of the pathogenesis of IPF. Idiopathic pulmonary fibrosis (IPF) is a non-neoplastic pulmonary disease, characterized by extracellular matrix deposition, myofibroblasts foci formation and alveolar epithelial cell hyperplasia In this study, we analyze global methylation patterns of IPF using human CpG island microarrays. In addition to compiling a DNA methylation profile that differentiates IPF patients from normal individuals, we compared this profile to that of lung adenocarcinoma patients. Our results reveal an altered DNA methylation pattern in IPF which shows great similarity to the methylation pattern of lung cancer. Our work is the first step in understanding the role of DNA methylation in the pathogenesis of IPF. Furthermore, the similarity of IPF with cancer may reveal common underlying molecular mechanisms and offer therapeutic options for IPF patients adopted from cancer biology 1b-T2b N0 M0Lung tissue samples were obtained through the University of Pittsburgh Health Sciences Tissue Bank . They included 12 frozen lung tissue samples from IPF patients, 10 frozen lung tissue samples from adenocarcinoma patients and 10 histological normal lung samples obtained from the same group of adenocarcinoma patients Table 1.Genomic DNA was extracted from 10 mg of frozen lung tissues using the DNeasy Blood & Tissue kit  following the manufacturer’s protocol. Five micrograms of genomic DNA were sonicated  to achieve fragment lengths between 300–600 bp. Methylated DNA was immunoprecipitated (IP) using 5-Methylcytidine monoclonal antibody  as instructed in the Agilent Microarray Analysis of Methylated DNA Immunoprecipitation protocol  For our analysis we only included probes with a hybridization Tm value between 79°C and 93°C as these show higher quality signal http://www.epidesigner.com/index.html)  Total RNA was extracted from frozen lung tissue with miRNeasy mini kit  following the manufacturer’s protocol http://linus.nci.nih.gov/BRB-ArrayTools.html). We controlled for multiple testing by setting the significance level at a False Discovery Rate (FDR) of less than 5% t test was applied to for the EpiTYPER MassArray and qRT-PCR to test significance of the results. Significance of overlap of differentially methylated islands (DMI) between IPF and Cancer samples and enrichment of DMIs in promoter regions was calculated using the hypergeometric distribution. Pathway analysis was performed using DAVID Bioinformatics Resources 6.7 http://www.ingenuity.com).Differentially methylated CpG islands were identified by analyzing the CpG Island Microarray data with the Class Comparison feature of BRB-ArrayTools 3.7.0 . 91.2% of the 625 differentially methylated CpG islands were located in intronic, exonic or and intergenic areas and only 8.8% in promoters. Considering that 10,923 of the 25,406 (43%) CpG islands in our study localize to promoters, this result indicates that a significantly larger than expected (p < 10–79) proportion of changes in methylation, when comparing IPF and control samples, occurs in regions that are not annotated as promoters in the current genome build.The 25,406 out of 27,639 human CpG islands that had an acceptable Tm (see methods) were analyzed using the Class Comparison algorithm from BRB Array Tool software package. 625 CpG islands were found to be differentially methylated in IPF lung tissue samples when compared to control lung tissue samples (Table S2 in Supporting Information). A Functional Annotation Clustering of these genes using DAVID Bioinformatics Resources 6.7 revealed that a significant number of them are involved in apoptosis, cell morphogenesis, the regulation of cellular biosynthetic processes and histone acetylation . We analyzed by qRT-PCR three genes localized in differentially regulated regions. Serine/Threonine Kinase 17b (STK17B) and Serine/Threonine Kinase 3 (STK3) are involved in apoptosis while histone cluster 1 H2ah (HIST1H2AH) is essential is nucleosome formation. All three transcripts showed increased levels of expression in IPF samples compared to controls but only STK17B and HIST1H2AH have a p-value <0.05 while in the case of STK3 the p-value is 0.07 . This overlap is highly significant, as the probability of such an overlap to occur in random is very low (p<10−256). 45% of the 402 overlapped CpG islands are located in intronic and intergenic areas, 6% in promoters and 49% in exons.To determine the similarity of IPF samples to lung cancer we performed Principal Component Analysis (PCA) and Class Comparison using BRB-Array Tools on microarray data from 12 IPF patients and the 10 lung cancer patients . PCA analysis demonstrated that IPF samples were positioned between the control and cancer samples suggesting that IPF samples had a methylation profile with partial similarity to both groups (Table S1 in Supporting Information). Although LINE-1 elements were found to be hypomethylated in the adenocarcinoma samples no significant change of the methylation levels was detected the in IPF samples  and Serine/Threonine Kinase 3 (STK3) because of their role in apoptosis et al compared IPF to cancer and described the pathogenic similarities between the two diseases. More specifically, they referred to common genetic and epigenetic alterations, uncontrolled proliferation, tissue invasion and perturbation of signal transduction pathways A remarkable finding of our study is the similarity in DNA methylation patterns between IPF and lung adenocarcinoma. Recently, Vancheri One of the major concerns in our global profiling approach is tissue heterogeneity. The IPF lungs contain mixed areas of normal tissue, myofibroblast foci and honeycombing To the best of our knowledge our study is the first one to describe global DNA methylation changes in IPF lungs. Taken together with the extensive changes in gene histology, gene expression and microRNA profiles our results highlight the profundity and complexity of events underlying the phenotypic changes in IPF and to some extent suggest that interfering with one pathway may not be sufficient to reverse these changes. The differentially methylated CpG islands we identified should be further studied as their regulation could provide insights about how genotype and the environment interact to determine the lung phenotype in IPF. Based on our results, we believe that epigenetic modifications play a key role in the pathogenesis of IPF and thus could serve as disease biomarkers and therapeutic targets.Table S1The sequence of EpiTYPER MassArray primers.(DOC)Click here for additional data file.Table S2Differentially methylated CpG islands distinguishing IPF from controls.(DOC)Click here for additional data file.Table S3Functional Analysis of the 55 Differentialy methylated promoters in IPF.(DOC)Click here for additional data file.Table S4Differentially methylated CpG islands overlapping between IPF and cancer.(DOC)Click here for additional data file.


Approximately 80 long interspersed element (LINE-1 or L1) copies are able to retrotranspose actively in the human genome, and these are termed retrotransposition-competent L1s. The 5′ untranslated region (UTR) of the human-specific L1 contains an internal promoter and several transcription factor binding sites. To better understand the effect of the L1 5′ UTR on the evolution of human-specific L1s, we examined this population of elements, focusing on the sequence diversity and accumulated substitutions within their 5′ UTRs. Using network analysis, we estimated the age of each L1 component . Through the comparison of the L1 components based on their estimated ages, we found that the 5′ UTR of human-specific L1s accumulates mutations at a faster rate than the other components. To further investigate the L1 5′ UTR, we examined the substitution frequency per nucleotide position among them. The results showed that the L1 5′ UTRs shared relatively conserved transcription factor binding sites, despite their high sequence diversity. Thus, we suggest that the high level of sequence diversity in the 5′ UTRs could be one of the factors controlling the number of retrotransposition-competent L1s in the human genome during the evolutionary battle between L1s and their host genomes. These en events –8 and arn events . Almost n events , 10–12. n events ; ORF1 enposition –16. The position , 18.Most previous studies of human-specific L1s focused on the 3′ UTRs rather than the 5′ UTRs because over 90% of L1 elements are 5′ truncated , 20, a rin vitro [The human genome contains ~2,000 copies of human-specific L1s , 24. Howin vitro . To explhttp://genome.ucsc.edu/cgi-bin/hgBlat) [A total of 1,835 human-specific L1 elements were previously identified . We manu/hgBlat) . The genIn a previous study of human-specific L1s, six L1 subfamilies were established based on the diagnostic mutations that are shared between all members of each subfamily . In thisFor the subfamily of L1Hs-preTa, we aligned the 5′ UTRs of all members and compared them with its consensus sequence using the biological sequence alignment editor BioEdit v.7.0 . For thehttp://line1.bioapps.biozentrum.uni-wuerzburg.de/l1xplorer.php) annotation tool [http://www.fluxus-engineering.com/sharenet.htm) software [All of the human-specific L1 elements were divided into their 5′ UTR, ORF1, ORF2, and 3′ UTR components using a combination of the L1Xplorer  between CpG contents and mutation rates of the L1 components was calculated. The two-tailed P value  was considered using the online freely available software, GraphPad QuickCalcs .Using an in-house Perl script, we counted the numbers of CpG and GpC sites on the consensus sequence of each L1 subfamily. To compare CpG contents among the four L1 components, we calculated the ratio of CpG to GpC for each component. Pearson's correlation coefficient  software [After an L1 element inserts into the human genome, mutations accumulate at different rates at each nucleotide position within the L1 element. These mutation rates are usually measured in substitutions (fixed mutations) per base pair per generation. A full-length human L1 is divided into four components: the 5′ UTR, ORF1, ORF2, and the 3′ UTR. Using the NETWORK 4.6  [r = 0.6665, P = 0.5356). Thus, we can state that the relatively high frequency of CpG sites within the L1 5′ UTR is not a major source of the higher mutation rates observed in this component.CpG dinucleotides occur at less than 10% of their expected frequency in the human genome because substitution at a CpG site, caused primarily by the methylation of cytosine, occurs 10- to 50-fold more frequently than other substitutions in primate genomes –38. In susc.edu) . Among tIn conclusion, the mutation rate of the L1 5′ UTR is higher than that of the other L1 components, ORF1, ORF2, and the 3′ UTR. However, the TF binding sites are relatively conserved among the human-specific L1 elements. We suspect that any host defense system(s) against L1 elements may be the cause of the higher mutation rate of the 5′ UTR and, in contrast, that L1 elements have kept the TF binding sites conserved against the host defense system(s) in order to survive; it could be the result of the evolutionary battle between L1s and their host. We believe that the increased frequency of substitutions within the 5′ UTRs could play a key role in regulating L1 retrotransposition activity and the number of retrotransposition-competent L1s in the human genome. However, not much is known about the factors causing the high level of sequence diversity in the L1 5′ UTRs. Although we suggest that the relationship between the L1 5′ UTR and other host factors, including the defense systems, causes the high mutation rate of the L1 5′ UTR, we could not rule out other possible factors such as the low fidelity of L1 reverse transcriptase. Thus, more research is needed to explore this intriguing possibility. Finding the factors that cause the increased mutation rates observed within L1 5′ UTRs will shed light on our understanding about L1 evolution and how the human genome may place controls on the retrotransposition rate of its resident L1 element population.Supplementary Table 1: The genomic information of 443 full-length L1 elements in the human genome was summarized.http://www.fluxus-engineering.com/sharenet.htm) software. For this calculation, we used 0.15% per site per myr as the nucleotide mutation rate.Supplementary Table 2: Age of chimpanzee-specific L1 elements was estimated by using the NETWORK 4.6 (Click here for additional data file.Click here for additional data file.


Saccharomyces cerevisiae, RPGs are unusual in that they are commonly present as two highly similar gene copies and in that they are over-represented among intron-containing genes. To investigate the role of introns in the regulation of RPG expression, we constructed 16 S. cerevisiae strains with precise deletions of RPG introns. We found that several yeast introns function to repress rather than to increase steady-state mRNA levels. Among these, the RPS9A and RPS9B introns were required for cross-regulation of the two paralogous gene copies, which is consistent with the duplication of an autoregulatory circuit. To test for similar intron function in animals, we performed an experimental test and comparative analyses for autoregulation among distantly related animal RPS9 orthologs. Overexpression of an exogenous RpS9 copy in Drosophila melanogaster S2 cells induced alternative splicing and degradation of the endogenous copy by nonsense-mediated decay (NMD). Also, analysis of expressed sequence tag data from distantly related animals, including Homo sapiens and Ciona intestinalis, revealed diverse alternatively-spliced RPS9 isoforms predicted to elicit NMD. We propose that multiple forms of splicing regulation among RPS9 orthologs from various eukaryotes operate analogously to translational repression of the alpha operon by S4, the distant prokaryotic ortholog. Thus, RPS9 orthologs appear to have independently evolved variations on a fundamental autoregulatory circuit.Ribosomal proteins are essential to life. While the functions of ribosomal protein-encoding genes (RPGs) are highly conserved, the evolution of their regulatory mechanisms is remarkably dynamic. In  Saccharomyces cerevisiae, which focused our attention on the introns of ribosomal protein genes. We then genetically deleted these introns to assess their function. Unlike mammalian introns, we found that yeast introns were not required for high levels of gene expression. Instead, particular introns  were required to fine-tune gene expression through autoregulation. Surprisingly, animal orthologs of these genes also use introns to autoregulate through multiple forms of alternative splicing. We speculate that the introns of ribosomal protein genes, in particular, readily evolve means for autoregulation to meet the demanding requirements of ribosomal protein genes to maintain tight control of gene expression.Eukaryotic genes are littered with non-coding intervening sequences, or introns, that must be precisely excised from a messenger RNA before it can be properly translated into protein. Despite their ubiquity, the evolution and function of introns remain poorly understood. Consequently, we cannot accurately predict the functions of individual introns in any organism. In this manuscript, we used a combination of comparative genomics and experimental tests to identify functional introns. First, we looked for signatures of selection to identify important introns in the model yeast  Saccharomyces cerevisiae. RPGs are highly over-represented among intron-containing genes (69% of RPGs contain introns compared to ∼5% of non-RPGs), which has been suggested to reflect ongoing selection for introns that provide one or more functions in gene expression S. cerevisiae and relatives). And second, the effect of intron loss on RPG expression remains uncertain.The evolution and function of spliceosomal introns are among the largest unsolved mysteries of eukaryotic genomes. Pronounced differences in intron evolution between lineages and between introns within the same lineage provide insight into 1) the selective and mutational forces governing intron evolution and 2) the potential roles of introns in gene function. Here we study the case of ribosomal protein genes (RPGs) in the model yeast S. cerevisiaeRPG expression is remarkable both in terms of synthesis rate and control RPL30 and RPS14B expression is achieved through the binding of their respective protein products to RNA structures within their own unspliced transcripts, thereby regulating splicing Escherichia coli are regulated by key ribosomal proteins in an analogous manner; for example, bacterial S4 directly binds its own mRNA to repress the translation of itself and three other RPGs S. cerevisiae RPGs contain introns, intron-dependent autoregulation may be more common than previously appreciated.In addition to the above, two other proposals predict that RPG introns function by providing an opportunity for splicing regulation. One possibility is that introns provide rapid regulation in response to environmental stress, as suggested by splicing inhibition of RPG pre-mRNAs in response to amino acid starvation S. cerevisiae strains harboring precise deletions of 16 RPG introns to distinguish between selective hypotheses. We found that RPG introns generally reduce gene expression, suggesting that RPG introns allow for splicing regulation rather than promoting high levels of expression. In particular, we identified intron-dependent cross-regulation between the RPS9A and RPS9B genes, which both encode ribosomal protein S9 (S9). Finally, overexpression of RpS9 in D. melanogaster S2 cells, and analysis of available EST sequences, suggest that autoregulation of RPS9 orthologs may involve different forms of splicing regulation between species, but also appears to be widespread across disparate lineages.We report the first direct tests of both the action and the source of selection on RPG introns. First, we used comparative genomics to show that RPG introns have been preferentially retained following whole genome duplication (WGD), indicating ongoing selection for retention of RPG introns. Second, we generated Candida albicans and S. cerevisiaeS. cerevisiae. To determine if RPGs have resisted intron loss compared to other genes since the divergence of C. albicans and S. cerevisiae  that were duplicated ∼100 million years ago by whole-genome duplication (WGD) S. cerevisiae, which has been exhaustively searched for introns Introns are over-represented in the RPGs of both S. cerevisiae duplicated gene pairs, we assigned the presence or absence of an intron in the hypothetical pre-WGD ancestral ortholog based on intron annotations and predictions from the genomes of the pre-WGD  species  and the genomes of the post-WGD species . A complete list of intron predictions and annotations can be found in S. cerevisiae were descended (S. cerevisiae WGD-derived gene pairs that have gained or lost an intron for each post-WGD gene pair (S. cerevisiae non-RPG pairs that appear to have independently lost introns from both gene copies after gene duplication. This was in addition to 14 non-RPG pairs in which one of two introns were lost (S. cerevisiae RPG pairs (S. cerevisiae during the last ∼100 million years.To better assess whether WGD-derived RPG pairs have been biased for either intron gain or loss (including losses in both gene copies), we reconstructed the hypothetical intron distribution of the pre-WGD ancestor that existed prior to the WGD event. For each of the 554 escended . Based oene pair . From thS. cerevisiae RPGs. For a given S. cerevisiae gene, we inferred that an intron was gained if introns were absent in both the pre-WGD ancestor and the majority of post-WGD orthologous gene pairs. Using this criterion, we did not infer intron gains in any of the S. cerevisiae RPGs. On the other hand, two introns in non-RPGs (i.e. USV1 and BMH2) have possibly been gained in the S. cerevisiae lineage . Each Δi mutant was created with a precise deletion of a single RPG intron, such that only an intronless copy of the gene remained at the endogenous locus  we used custom splicing-sensitive microarrays designed to detect pre-, mature, and total mRNA species  to inhibit splicing and to cause rapid degradation RPS14B intron led to a substantial increase in its expression compared to the other genes on the microarray . Our microarray experiments revealed that RPS9A and RPS9B Δi mutations increased the expression of the intronless genes , suggesting that these genes cross-regulate.We also sought to determine if any of the deleted introns were required for splicing regulation. As controls, we deleted the introns of pression  “down”. ss genes . We hypoRPS9A and RPS9B expression by RT-qPCR. Importantly, we designed at least one qPCR primer to the 3′UTR in an effort to maximize specificity and to minimize artifacts caused by primer cross-hybridization to the other gene copy. As expected, qPCR measurements validated our microarray results for both RPS9A and RPS9B genes in the rps9bΔi and rps9bΔi mutants  in RPS9A expression and a modest decrease (<2-fold of wild-type) in RPS9B expression  in RPS9B expression and a substantial decrease (>8-fold of wild-type) in RPS9A expression  deletion of both the RPS9A and RPS9B introns should eliminate cross-regulation, and therefore, derepress both gene copies and 2) the wild-type gene copy should compensate for a derepressed copy by an equal and opposite number of transcripts. First, to determine if repression of RPS9A expression in the rps9bΔi mutant required the RPS9A intron (and vice versa), we created a double rps9a/bΔi mutant and tested the effect on expression by RT-qPCR. As predicted, both RPS9A and RPS9B were derepressed in the rps9a/bΔi mutant  from a published RNA-seq data set from a wild-type strain rps9aΔi mutant, a substantial relative increase in RPS9A expression mutant was nearly equally compensated by a modest relative decrease in RPS9B expression, such that the total number of transcripts encoding S9 was nearly unchanged : a process in which the synthesis of productively-spliced mRNA is repressed in favor of unproductive mRNA isoforms encoding premature termination codons (PTC+) RpS9 PTC+ mRNA isoform was recently identified in Drosophila melanogasterRPS9 orthologs autoregulate in a manner analogous to RPS9A and RPS9B cross-regulation.Reminiscent of the cross-regulation between D. melanogaster RpS9 expression is regulated in response to excess protein production by alternative splicing coupled to NMD. Therefore, we predicted that increased RpS9 expression would result in increased abundance of the PTC+ mRNA isoform. To test this hypothesis, we measured the affect of exogenous RpS9 overexpression and NMD inhibition on alternative splicing of RpS9 messages using RT-qPCR primer sets specific to endogenous RpS9 mRNA isoforms , requires an RNA structure to autoregulate by translational repression. Therefore, we predicted that RPS9 orthologs would be associated with alternatively-spliced mRNA isoforms, conserved RNA structures, and PTCs. To identify such messages, we summarized expressed sequence tags (ESTs) data from diverse animals. Indeed, EST coverage extends outside exons and into introns, which support the existence of rare unspliced or alternatively-spliced transcripts (<5% maximum coverage)  reveal cassette exons that introduce PTCs from the last canonical intron , specifically within the last intron of mammalian, drosophilid, and teleost RPS9 orthologs, each overlapping with PTC positions  the mechanisms of intron loss and/or 2) selection to keep important introns. In addition to previously-proposed functions of RPG introns (see below), several lines of evidence suggest that the conservation of RPG introns is not merely a function of mutation rates. Reverse transcription-mediated intron loss is expected to preferentially remove 3′ end biased introns from highly-expressed genes S. cerevisiae intron losses observed here are not 3′ end biased; in fact, several introns were lost from the 5′ UTR . Lastly, at least 21 RPG introns that are present in both the Lachantea and Saccharomyces clades appear to have been lost from Z. rouxii, indicating that species-specific RPG intron losses can occur, but have not done so in the lineage leading to S. cerevisiae  are regulated by splicing in both yeast and mammals  exemplify deep homology, where recent evolutionary innovations overlay a shared “genetic toolkit” S. cerevisiae may be different elaborations on this autoregulatory circuit, perhaps mediated by different RNA structures within introns  on 7/20/2011. WGD-derived gene pairs  were obtained from the Yeast Gene Order Browser (http://wolfe.gen.tcd.ie/ygob/) http://www.r-project.org/) were used to scan 800 bp upstream and 100 bp downstream of the ORF start site with a regular expression that recognizes >90% of S. cerevisiae introns by identifying the most common splice sites to minimize false positive matches. The regular expression matches sequences meeting the following criteria in order: 1) any one of the 4 most common of 5′ SSs, 2) an S1 length of at least 30, 3) any one of the 5 most common branchsites, 4) an S2 length between 1 and 50, and 5) any one of the 3 most common 3′ SS trinucleotides, which was formalized as: “(gtatgt|gtacgt|gtaagt|gtatga).{30,}?(tactaac|gactaac|aactaac|tgctaac|cactaac).{1,50}?[tca]ag”. The pre-WGD ancestor was inferred to contain an intron if the majority of available outgroup pre-WGD species orthologs  and 1) the Z. rouxii ortholog had an intron or 2) the majority of post-WGD species intron  gene pairs had at least one intron. In this manner, we distinguished independent intron gains and losses in Z. rouxii from intron gains and losses immediately after the WGD event.To estimate the propensity for intron loss among RPGs and non-RPGs, we compared annotated Intron deletion mutants were generated by a replacement strategy similar to a previously-described method for intron deletion Routine passaging of S2 cell cultures and RNAi depletion was performed as described S. cerevisiae intron deletion strains, 15 ml cultures of mutant and wild-type yeast were grown in parallel at 30°C in rich medium supplemented with 2% glucose to an optical density between A600 = 0.5 and 0.7. For microarray hybridization, RNA was isolated by acid-phenol extraction and converted to cDNA as described D. melanogaster S2 cell, 725 µl cultures of S2 cells (UCSF cell culture facility) were grown in 24-well plates at 25°C in Schneider's Drosophila Medium (Gibco) supplemented with 10% fetal bovine serum (UCSF) to a count of ∼5E6 cells. RNA was extracted with 1 ml TriZol (Invitrogen) according to manufacturer's instructions. After RNA isolation, cDNA was prepared with SuperScript III (Invitogen) and random priming according to manufacturer's instructions. Prepared cDNA was diluted at least 10-fold before use in qPCR.To analyze the expression of genes in Splicing-sensitive microarrays were constructed and performed as described S. cerevisiae or D. melanogaster genomic sequence obtained from the UCSC Genome Browser  4)2SO4 buffer (Fermentas), 0.5 µM primer, 1.5 mM MgCl2, 1.25 Units Dynazyme II (Finnzymes), and Sybr Green I fluorescent dye (Sigma). Fluorescence was measured on a BioRad Opticon machine using standard cycling conditions . Biological replicate qPCR values were determined as the median of technical replicates. For each of 3 biological replicates, target gene values (e.g. RPS9A) were divided by reference gene values (e.g. SCR1) before log transformation. Plots were generated using the R package ggplot2 Quantitative PCR primers  were desBranchiostoma floridae (braFlo1); Ciona intestinalis (Ci2); Danio rerio (danRer7); Drosophila melanogaster (dm3), Oryzias latipes (oryLat2); Petromyzon marinus (petMar1); Xenopus tropicalis (xenTro2); Rattus norvegicus (rn4); Mus musculus (mm9); Homo sapiens (hg19).To assess EST coverage and splicing, we obtained genomic coordinates corresponding to GenBank ESTs from the UCSC Genome Browser ‘spliced EST’ track, which span at least one canonical intron of at least 32 bases Figure S1RPS9 orthologs from 10 animals. EST summaries of RPS9 orthologs from 10 animal species illustrated as in Comparison of spliced isoforms by EST analysis of (PDF)Click here for additional data file.Figure S2RPS9 orthologs. Nucleotide alignment of genes encoding ribosomal protein S9 from L. waltii, L. thermotolerans, L. kluyveri, E. gossypii, K. lactis, Z. rouxii, S. cerevisiae, S. bayanus, C. glabrata, N. castellii and V. polyspora. Note that all genes contain an intron with regions of identical sequences (black shading). Positions of compensatory base pair changes supporting RNA stems shown in Conserved intronic regions among yeast (PDF)Click here for additional data file.Figure S3RPL30 and RPS14 does not reveal conserved alternatively-spliced isoforms. EST summaries of RPL30 and RPS14 orthologs from human, mouse, and rat illustrated as in EST analysis of mammalian (PDF)Click here for additional data file.Figure S4RPS9A and RPS9B introns. Conserved elements in the RPS9A and RPS9B introns are associated with a putative pseudoknot structure near the 5′ splice site. A) An illustration of the S. cerevisiae RPS9A gene model and nucleotide conservation among closely related yeasts (from the UCSC Genome Browser). Putative RNA stems (predicted by the RNAz program http://pseudoviewer.inha.ac.kr/).Putative RNA structure within the (PDF)Click here for additional data file.Table S1Intron annotations and predictions.(PDF)Click here for additional data file.Table S2Primers.(PDF)Click here for additional data file.


Sarcoidosis is a chronic disorder that can virtually affect any organ system in the body. Histologically, it is characterized by the presence of T lymphocytes, mononuclear phagocytes, and noncaseating granulomas. Most commonly affected are the intrathoracic structures, with 90% of the reported cases involving the lungs. Esophageal involvement in sarcoidosis is extremely rare. Dysphagia is the most common presentation in these patients and can be attributed to various mechanisms such as direct esophageal wall infiltration, extrinsic compression, cranial neuropathy, and brainstem involvement. A thorough online literature review revealed only 23 reported cases of esophageal involvement in sarcoidosis. This paper reviews these reported cases in detail along with newer diagnostic and treatment options, including direction of future therapy. Sarcoidosis is an inflammatory, granulomatous, multisystem disorder of unclear etiology . The lunSarcoidosis is a global disease with variable incidences, presentation, and prognosis. It has the highest incidence in the United States and Sweden . In the Gastrointestinal tract involvement in sarcoidosis is very rare. Elaborate postmortem studies of patients with sarcoidosis, published between 1949 and 1963, did not find any evidence of GI tract involvement –13. HoweEsophageal involvement is an extremely rare occurrence in sarcoidosis. A review of the literature revealed only 23 published cases of esophageal involvement in sarcoidosis to date –42. The Mycobacterium tuberculosis katG and M. tuberculosis topoisomerase [P. acnes have been frequently detected in patients with sarcoidosis [Cytomegalovirus, Mycoplasma, Herpes virus and Borrelia Burgdoferi [The exact causes of sarcoidosis remain unknown. However, various infectious and environmental antigens have been suggested as triggers, causing sarcoid to develop in a genetically susceptible individual. The immune response in sarcoid is similar to those present in other granulomatous diseases caused by a known antigen, such as tuberculosis and berylliosis. There have been various reports of community outbreaks and clustering of cases in time and space which has led to speculation and suggestions of a possible environmental antigen or even a person to person transmission of sarcoid. There have been reports of increased disease incidence and clustering in certain occupations such as nurses and firefighters , 43. Occsomerase . Similarcoidosis . This wacoidosis . Other irgdoferi , 49. β), interleukin- (IL-) 1α and C-C chemokine receptors. The increased levels of angiotensin converting enzyme (ACE) in some patients with sarcoidosis has led to interest and enthusiasm in evaluation of ACE gene polymorphisms. However, most reviews have found no association between these polymorphisms with sarcoidosis [The increased occurrence of sarcoidosis among families and an increased incidence specifically among monozygotic twins had led to initial suggestions that genetic susceptibility plays an important role in sarcoidosis. Compared to Caucasians families where only 5% had more than one affected individual, the rates in African American families were as high as 19% . Studiescoidosis , 51, 52.The characteristic finding of sarcoidosis is the presence of noncaseating granulomas. The granuloma is comprised of mainly lymphocytes, epitheloid cells, macrophages, and fibroblasts in varying proportions. Some of the macrophages undergo differentiation to form epitheloid cells, mature, lose some of its phagocytic activity, and fuse to form multinucleated giant cells. During the early, acute phase, the core of the granuloma consists of mononuclear phagocytes in different stages of maturation and differentiation. This is surrounded by mainly CD4+ T cells and a few CD8+ T cells and B cells. CD8+ cells and fibroblasts become increased in number during the latter stages, as the granulomas become less active. Granulomas can also be identified in a number of other infectious and noninfectious states. Granulomas found in sarcoidosis are classically nonnecrotizing, tense and usually track along the lymphatics. Granulomas found in hypersensitivity pneumonitis or extrinsic allergic alveolitis are generally nonnecrotizing too, but are loosely formed and track along the airways. Granulomas seen in tuberculosis and histoplasmosis, however, are classically necrotizing tense and are distributed in no particular pattern. The lesions in berylliosis are very similar to those found in sarcoidosis, and therefore effort must be made to inquire about occupational exposure to beryllium and its various compounds .γ (IFN-γ). This further exaggerates the immune response and induces the proliferation and chemotaxis of T lymphocytes. Blood monocytes that arrive then undergo differentiation into exudates macrophages which in turn releases factors such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which enhances the formation of a granuloma. These cells then further undergo organization and ultimately form noncaseating granulomas. It has been postulated that increased levels of transforming growth factor-β (TGF-β), insulin-like growth factor-1, and various changes in cytokine production  that now favor a Th2 cell pattern are responsible for progression to a fibrotic change in the granuloma [Macrophages and dendritic cells present the antigens through the framework of the major histocompatibility complex (MHC) class II alleles to the T cells. They also secrete cytokines interleukin-12 (IL-12) which stimulates the maturation of CD4+ T lymphocytes to Th1 cells and interleukin-15 (IL-15) which promotes T lymphocyte propagation. CD4+ T lymphocytes which are the one of the primary cells involved in immunopathogenesis of sarcoidosis releases interleukin-2 (IL-2) and interferon-ranuloma , 54. A systematic review of the literature using the keywords “gastrointestinal sarcoidosis,” “esophageal sarcoidosis,” and “GI sarcoidosis” was done on Medline/Pubmed, Google Scholar, and the directory of Open access journals and by reviewing references of earlier published case reports. Our search yielded twenty-three documented cases of esophageal involvement in sarcoidosis. A summary of these reported cases can be found in Dysphagia has been reported to be the most common symptom in patients with esophageal sarcoidosis . This clSuperficial involvement of the mucosa may manifest macroscopically as mucosal hyperemia, discrete plaque-like or nodular lesions. Murdock and Jacob  presenteMyopathic involvement in esophageal sarcoidosis occurs due to infiltration of the skeletal muscles of the esophagus and posterior pharynx. Siegel et al.  reportedDirect involvement of the enteric nervous plexus can cause dysphagia, and the clinical picture can mimic that of achalasia. Dufresne et al.  describeDysphagia as a consequence of extrinsic compression from enlarged mediastinal lymph nodes due to sarcoidosis has also been described. Cook et al.  reportedThe esophageal involvement in sarcoidosis can be classified based upon two criteria: the level of involvement and the layer of involvement . The lowDiagnosis of sarcoidosis hinges upon establishing a compatible clinical and histopathologic picture whilst ruling out other granulomatous diseases. A detailed history including particulars about environmental and occupational exposure is key to identifying other granulomatous diseases. A detailed physical exam aids in assessing the extent of involvement of other organ systems. A baseline slit lamp examination to diagnose subclinical uveitis and measurement of 24 hour urine calcium to detect subclinical hypercalciura, are also recommended in all patients. Cardiac involvement, though rare, can cause life-threatening arrhythmias and therefore electrocardiograms are recommended at baseline and at frequent intervals  found thThere have been no clinical trials comparing the efficacy of the different treatment modalities for the treatment of esophageal sarcoidosis given the rarity of the disease. Dietary modifications, proton pump inhibitors, and surgical and corticosteroid therapy have been used to treat esophageal sarcoidosis. The role of corticosteroids in the treatment of gastrointestinal sarcoidosis is not clear. However, considering the presence of systemic involvement in almost all cases of esophageal sarcoidosis and the observation that esophageal inflammation secondary to granulomatous inflammation seems to respond to corticosteroids, oral steroids remain the first-line therapy . It has α, and TNF, it also inhibits the formation of proinflammatory enzymes including collagenase and plasminogen activator. Moreover, corticosteroids have a negative impact on lymphocyte proliferation and delayed hypersensitivity reactions. The goal of steroid therapy in systemic sarcoidosis is to relieve symptoms thus improving quality of life and to halt the progression of disease and irreversible organ damage [Besides inhibiting the production of inflammatory cytokines such as IL-1, IL-2, IFN-n damage . There hn damage , 55, 66.The aim of surgical treatment is to provide symptomatic relief, eradicate outflow tract obstruction, and possibly prevent acid reflux. Cricopharyngeal myotomy can be used for the treatment of both oropharyngeal dysphagia and hypopharyngeal (Zenker's) diverticulum. Cricopharyngeal myotomy was first performed by Kaplan et al. in 1951 for the treatment of post-poliomyelitis dysphagia and it has since then been increasingly used for dysphagia due to various causes . The proEsophageal myotomy to relieve achalasia was first reported by Ernest Heller in 1913 wherein both the anterior and posterior esophageal sphincters were cut. A modification of this technique involves only the dissection of the anterior muscle fibers and this has become the standard surgical procedure . HoweverBotulinum toxin is an irreversible inhibitor of acetylcholine release from presynaptic cholinergic terminals and this helps lower the lower esophageal sphincter tone. However, since new axon growth and regeneration counteracts this effect over time, treatment needs to be repeated at regular intervals. Multiple injections into the lower esophageal sphincter as a result of repeat treatment can cause local inflammation and fibrosis, further limiting the usefulness of this therapy. This option is thus reserved for older patients or those who are not ideal candidates for either surgical or corticosteroid therapy.Due to the rarity of esophageal involvement in sarcoidosis, the role of immunosuppressive therapy has not been defined. However, their use must be reserved for those patients who experience side effects due to systemic steroid therapy or in those with worsening of symptoms despite corticosteroids. Methotrexate is the most commonly used immunosuppressive agent in systemic sarcoidosis. Methotrexate is believed to decrease the antigen stimulated proliferation of lymphocytes and exerts its anti-inflammatory through adenosine accumulation , 71. Theα therapies may be valuable agents in the treatment of sarcoidosis [α), etanercept (soluble TNF receptor that binds to TNF in circulation), and adalimumab  are agents that are rarely used in the treatment of sarcoidosis. Few clinical series and trials have shown benefit of infliximab in the treatment of refractory sarcoidosis, including extrapulmonary manifestations [α agents has been described in the literature [As mentioned earlier, TNF production is a fundamental part of the immunologic process of sarcoidosis. A recent study found TNF to be secreted at higher levels in a subset of patients with refractory sarcoidosis. These observations have led to the logical idea that anti-TNF-coidosis . Thalidostations . Given tterature . α drugs in esophageal sarcoidosis will need further research and intense scrutiny. Clinicians must be cognizant of this rare but clinically important disease process and an interdisciplinary team approach is essential for a favorable outcome. Gastrointestinal sarcoidosis is an extremely infrequent disease with clinically significant involvement seen in less than 1% of patients with sarcoidosis. The diagnosis of esophageal sarcoidosis can be challenging, not only because of the rarity of the disease but also because of the need for histopathologic evidence and the necessity to exclude other granulomatous diseases. It is likely that esophageal sarcoidosis is under-diagnosed due to the slow progression of the disease and difficulties in diagnosing the same. Upper endoscopy, esophageal manometry and endoscopic ultrasound are some valuable tools that can aid in timely diagnosis and therapeutic intervention. Corticosteroid therapy and various surgical techniques are most commonly used to relive symptoms of dysphagia secondary to esophageal involvement. The routine use of disease modifying agents including anti-TNF-


Shigella T3SS substrate, prevents premature effector secretion. Here, we examine how the different parts of T3SSs work together to allow orderly secretion. We show that T3SS assembly and needle tip composition are not altered in an mxiC mutant. We find that MxiC not only represses effector secretion but that it is also required for translocator release. We provide genetic evidence that MxiC acts downstream of the tip complex and then the needle during secretion activation. Finally, we show that the needle controls MxiC release. Therefore, for the first time, our data allow us to propose a model of secretion activation that goes from the tip complex to cytoplasmic MxiC via the needle.Type III secretion systems (T3SSs) are key determinants of virulence in many Gram-negative bacteria, including animal and plant pathogens. They inject ‘effector’ proteins through a ‘needle’ protruding from the bacterial surface directly into eukaryotic cells after assembly of a ‘translocator’ pore in the host plasma membrane. Secretion is a tightly regulated process, which is blocked until physical contact with a host cell takes place. Host cell sensing occurs through a distal needle ‘tip complex’ and translocators are secreted before effectors. MxiC, a  Type III secretion systems (T3SSs) are key determinants of virulence in many Gram-negative bacteria, including animal and plant pathogens. They are protein transport devices used to inject ‘effector’ proteins into the plasma membrane or cytoplasm of host cells to manipulate cell processes. Effectors can modulate intracellular trafficking, induce apoptosis, provoke reorganization of the cytoskeleton and manipulate the immune response . In geneShigella flexneri, the causative agent of human bacillary dysentery, uses a T3SS to inject effector proteins that will allow invasion and dissemination through the colonic epithelium. In order to inject effector proteins into host cells, three proteins known as the ‘translocators’ , are required. IpaB and IpaC will assemble a pore in the host cell membrane, through which other virulence effector proteins may then be translocated , encoding proteins involved in later stages of infection, is controlled by the T3SS activity. Indeed, it is prevented by OspD1, an early secreted effector that represses the transcriptional activator MxiE (virA) is detected when the T3SS is not active but increases after secretion is activated , ipaB and ipaD mutations. We also show that when compared with wild-type, the mxiC mutant has a similar needle tip composition. Taken together, our data lead to a novel working model of secretion activation that, for the first time, goes from the tip complex to cytoplasmic MxiC via the needle. Beyond providing important and generalizable information on regulation of T3SS activation, this work begins to establish a new paradigm for how the functional state of macromolecular machines is internally sensed and regulated.We have further characterized an Shigella T3SS displays three different functional states  when compared with the wild-type strain , termed ‘effector mutants’ since they secrete translocators after CR addition, yet are concomitantly unable to secrete effector proteins (mxiHK69A and mxiHR83A. Both strains are unable to secrete MxiC after CR addition (The needle and its tip are involved in the control of T3SS activity as some mutants in the needle subunit (MxiH) show an altered tip complex and secretion phenotype  mutant should show as weak an induction of translocators secretion as the single mxiC mutant. We constructed an mxiC mxiH mutant, complemented it with mxiHK69A or mxiHR83A and analysed the secretion profile of these strains. As expected, the mxiC mxiH double mutant did not secrete any of the translocator or effector proteins (data not shown). We found that mxiC mxiHK69A  indeed secretes early effectors constitutively  showed as weak an induction of translocators secretion as the single mxiC mutant  that behaves as a constitutive secretor, yet remains invasive and recruits IpaC to the needle tip  proteins present in needles.As secretion was altered in an ern blot . We founmxiC mutant could have a defect in T3SS assembly or number, cultures from the wild-type, the mxiC mutant and the mxiC complemented strains were osmotically shocked to produce ‘ghost’ cells where secretons are observable at the cell periphery , as previously described , MxiC must be acting from inside the cell at this point. Therefore, we presume that the repression mechanism is also intracellular. LcrV, the Yersinia IpaD homologue, regulates secretion through interaction with the negative regulator protein LcrG, its intracytoplasmic chaperone. However, unlike IpaD, LcrV acts as a positive regulator, as an lcrV mutant shows a defect in effector secretion  presence of MxiHK69A prevents MxiC secretion and as a consequence effector secretion  at 37°C with the appropriate antibiotics at the following final concentrations: ampicillin 100 µg ml−1, kanamycin 50 µg ml−1, tetracycline 15 µg ml−1.mxiC mutant was generated by using the Lambda Red system as described by mxiC gene, a tetracycline resistance cassette with 50 pb flanking regions homologous to the mxiC gene was amplified from strain TH2788 , was grown at 30°C to an OD600 of 0.2 and then induced by adding arabinose to a final concentration of 0.2% during approximately 2 h. When cells reached an OD600 of ∼ 0.6, they were made electrocompetent by concentrating them 100 times and washing them 3 times with ice-cold water. They were electroporated with 1 µg of the purified PCR product described above. Mutants where the mxiC gene has been replaced by the tetracycline cassette were selected on plates with tetracycline. The same procedure was used to inactivate mxiC in strains SF620 (ipaB) and SH116 (mxiH) giving rise to strains mxiC ipaB and mxiC mxiH respectively. To construct strain ipaB mxiH, the ipaB gene was exchanged for a tetracycline cassette in the single mutant strain SH116 (mxiH) using the same method. All gene replacements were confirmed by sequencing.The n TH2788 . ShigellmxiC mutant, mxiC was amplified by PCR from the M90T virulence plasmid pWR100 using primers mxiC_SalI and mxiC_BHI (ipaB and mxiH or mxiHK69A are derivatives of pDR1 that contains ipaB cloned into pUC19 (mxiH or mxiHK69A were amplified by PCR from pRKmxiH or pRKmxiHK69A (ipaB gene). All plasmids were verified by sequencing.To complement the mxiC_BHI  and clonShigella flexneri strains were grown at 37°C until exponential growth (OD600 ≈ 1) was reached. Samples of the cultures, representing the total protein fraction, were denatured in Laemmli sample buffer, separated by SDS-PAGE and used for Western blot analysis.Shigella flexneri strains were grown overnight at 37°C. Bacteria were collected by centrifugation at 15 000 g for 10 min at 4°C. The ‘stationary phase’ supernatants were denatured in Laemmli sample buffer, subjected to SDS gel electrophoresis and Silver-stained .600 ≈ 1) were resuspended at OD600 = 5 in phosphate-buffered saline. Congo red was added  to induce T3SS activity. After incubation at 37°C for 15 min, the samples were centrifuged at 15 000 g for 10 min at 4°C and the supernatants were denatured, separated by SDS-PAGE and Silver-stained or used for Western blot analysis.Bacteria collected during exponential growth  used for Ipa protein detection have been described previously and were generous gifts from Armelle Phalipon  and Kirsten Niebuhr (αIpgD) , the mou600 of 1 using glass beads as described previously  and cells were lysed by two passages through a French Press at 10 000 Pa and a few crystals of DNase I were added to reduce sample viscosity. Unbroken cells were removed by several rounds of centrifugation (10 min at 10 000 g), until no further pellet was obtained. Cell lysates (0.66 ml) were added to a TLS 55 Ultraclear centrifuge tube  containing 20% w/w sucrose solution (1.54 ml). Membranes were pelleted by ultracentrifugation at 200 000 g for 1 h at 4°C in an Optima TLX Ultracentrifuge (Beckman). Three fractions were collected, the first two by always rotating the collection pipette slowly around the tube's edge at the solution's meniscus: the top, cytosol fraction , the middle, sucrose cushion fraction and the bottom, membrane pellet, which was first carefully washed with water and then resuspended in 0.6 ml Tris buffer. The sucrose fraction was precipitated with 10% trichloroacetic acid according to conventional procedures and finally resuspended in 0.6 ml Tris buffer. Five microlitres of each of the samples was separated by SDS-PAGE and used for Western blot analysis.Around 400 ml of bacteria was grown to an OD