We report a 52-year-old patient who developed B-cell non-Hodgkin’s lymphoma subsequent to sarcoidosis. Sarcoidosis was diagnosed 16 years ago and remained asymptomatic for 14 years after steroid treatment. She presented with new symptoms of arthralgia, photosensitivity, butterfly erythema, autoimmune antibodies  associated with progression of the known left upper lobe lesion on the chest X-ray suggesting primary autoimmune disease (systemic lupus erythematosus). As steroid treatment was not effective, we started bolus cyclophosphamide therapy after which progression was seen on the chest X-ray. Computed tomography (CT)-guided needle biopsy confirmed malignancy of indefinable origin. Despite of the well-known fluorodeoxyglucose (FDG) avidity in active sarcoidosis, a FDG-positron emission tomography (PET) scan was performed to stage the primary tumour. Intensive FDG uptake was detected in the affected lung segment, with moderate uptake in mediastinal lymph nodes. The patient underwent left upper lobectomy. The histology showed pulmonary mucosa-associated lymphoma (bronchus-associated lymphoid tissue (BALT) lymphoma) in the lung tissue, while only sarcoidosis was present in the mediastinal lymph nodes. Bone marrow biopsy was negative.The association between sarcoidosis and lymphoma is known as sarcoidosis lymphoma syndrome, which is a rare disease. PET-CT was helpful in the differentiation of sarcoidosis and malignancy in this patient. It is important to be aware of the risk of lymphoma in sarcoidosis and FDG-PET, used for adequate purpose, can help the diagnosis. Sarcoidosis is a chronic granulomatous inflammatory multisystem disorder of unknown origin. The association between sarcoidosis and malignancy - particularly lymphoproliferative disease, such as non-Hodgkin’s lymphoma - has been previously described -3. In sa2 monthly) therapy was instituted. Repeated chest X-ray showed rapid and significant worsening of the left upper lobe lesion following the first dose  showed bilateral hilar lymphadenopathy and lung parenchyma involvement. Histology confirmed sarcoidosis, which was treated with systemic steroids for six months, after which persisting diabetes mellitus developed. After 14 asymptomatic years without immunosuppressive therapy, she was referred to our department with symptoms of dry cough, snoring and mild anaemia. On the chest X-ray, progression of the known left upper lobe lesion was noted  lymphoma) in the left upper lobe lung tissue, while only sarcoidosis was present in the mediastinal lymph nodes. Immunophenotyping demonstrated a B cell lineage phenotype: CD20, BCL2 and lambda light chain positivity on the cell surface, with CD10 and CD5 negativity Figure  and 7.Sarcoidosis is a multisystem granulomatous disorder of uncertain origin. The diagnosis of sarcoidosis is based on clinical, radiological, biochemical and histological findings. These findings are erythema nodosum, lymphopenia, hypergammaglobulinemia, anergy, hypercalcemia and elevated angiotensin-converting enzyme. The non-caseating granulomas are B-cell negative . The resBrincker and Wilbek have matched all patients who had been diagnosed with respiratory sarcoidosis during the period 1962 to 1971 against the data of the Cancer Registry. About 1.5 times higher incidence of cancer was seen in sarcoidosis patients. Lung cancer occurred 3 times and malignant lymphoma 11.5 times more frequently than in the control population . In a reet al. described six patients who developed malignancy first, followed by the diagnosis of sarcoidosis. The median interval between the diagnoses of the two diseases was only nine months [et al. reported two cases, where NHL was identified and treated for 3 and 10 months before the diagnosis of sarcoidosis. They proposed that sarcoidosis had been triggered by immunological disturbance induced by chemotherapy or associated with the lymphoma. They supposed an infectious agent, which could have spread due to the immunosuppression (caused by chemotherapy) resulting in granuloma formation. Alternatively antineoplastic therapy might have reduced suppressor T cells leading to lymphocyte activation observed in sarcoidosis [et al. reported a patient with osteosarcoma who developed granulomatous lymphadenopathy and multiple pulmonary nodules confirmed as sarcoidosis following cessation of treatment. They suggested that immunosuppression inhibited the development of sarcoidosis, which became symptomatic when chemotherapy was terminated [There are many theories explaining the possible relationship of this association. The changes in the number and the functions of immune cells in sarcoidosis are well described, suggesting dysfunction in the immunoregulatory pathways leading to granuloma formation. A clone may escape in an environment with defective T suppressor cells regulation resulting in the development of lymphoid malignancy. In this context, chronic sarcoidosis represents a more prolonged and more severe form of the disease. Steroid treatment, by further compromising the immune system, might be an additional predisposing factor . There ae months . There acoidosis . Sybert FDG-PET is a sensitive method for the staging of several malignancies, the underlying biological principle is based on the Warburg effect . FDG is et al. found that BALT lymphomas show heterogeneous but identifiable FDG uptake on PET scans [In a study with 21 patients, Bae ET scans . They anET scans .According to the current literature, our patient had chronic sarcoidosis and had been treated with systemic steroids. The development of autoimmunity (SLE) and its treatment with cyclophosphamide induced changes in the slow growing tumour. This is in line with the theory that abnormal immune function contributes to the development of LD, and that immunosuppressive therapy may enhance it ,17. The According to previous observations, sarcoidosis predisposes for lymphoid malignancies. Sarcoidosis and lymphoma both can cause increased FDG uptake in mediastinal lymph nodes, thus FDG-PET-CT cannot prevent the need for histological verification . In our Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.ANA: Antinuclear antibodies; BALT: Bronchus associated lymphoid tissue; CO: Carbon-monoxide; CT: Computed tomography; FDG: Fluorodeoxyglucose; LD: Lymphoproliferative disease; MALT: Mucosa associated lymphoid tissue; NHL: Non-Hodgkin’s lymphoma; PET: Positron emission tomography; SLE: Systemic lupus erythematosus.The authors have no competing interests to declare.AK wrote the paper. NE and LT have been involved in drafting the manuscript. GYL and VM drafted the manuscript and revised it critically for important intellectual content. ACS performed the lobectomy. JCS carried out the histological examination and immunophenotyping. All authors read and approved the final manuscript.


Graphene oxide composites with photocatalysts may exhibit better properties than pure photocatalysts via improvement of their textural and electronic properties.2-Graphene Oxide (TiO2 - GO) nanocomposite was prepared by thermal hydrolysis of suspension with graphene oxide (GO) nanosheets and titania peroxo-complex. The characterization of graphene oxide nanosheets was provided by using an atomic force microscope and Raman spectroscopy. The prepared nanocomposites samples were characterized by Brunauer–Emmett–Teller surface area and Barrett–Joiner–Halenda porosity, X-ray Diffraction, Infrared Spectroscopy, Raman Spectroscopy and Transmission Electron Microscopy. UV/VIS diffuse reflectance spectroscopy was employed to estimate band-gap energies. From the TiO2 - GO samples, a 300 μm thin layer on a piece of glass 10×15 cm was created. The photocatalytic activity of the prepared layers was assessed from the kinetics of the photocatalytic degradation of butane in the gas phase.TiO-1), while sample labeled TiGO_075 (k = 0.00774 h-1) demonstrated the best activity under visible light.The best photocatalytic activity under UV was observed for sample denoted TiGO_100 (k = 0.03012 h It has been well documented that GO is heavily oxygenated graphene that is readily exfoliated in water to yield stable dispersions consisting mostly of single-layer sheets. The use of graphene oxide as the nanoscale substrates for the formation of nonocomposites with metal oxides is widely explored due to an idea to obtain a hybrid which could be combined both properties of GO as fascinating paper-shape material and the features of single nano-sized metal oxide particles.Traditionally, TiO2-GO) and titanium oxide nanoparticle-reduced graphene oxide (TiO2-RGO) composites were early provided and emphasized on the different synthetic strategies for the preparation of graphene-TiO2 materials. For example, TiO2-GO materials have been prepared via the hydrolysis of TiF4 at 60°C for 24 h in the presence of an aqueous dispersion of graphene oxide (GO). The reaction proceeded to yield an insoluble material that is composed of TiO2 and GO 2+ induced by the hydrolysis of Ti(SO4)2 diffused into graphene oxide interlayer by electrostatic attraction [TiOfate TiSO2 as initRecently, Min et al.  presente2 nanocomposites was published [2 to CH4. Another novel method [2 composites as a highly efficient photocatalyst by in situ depositing TiO2 nanoparticles on graphene oxide nano-sheets by a liquid phase deposition, followed by a calcination treatment at 200°C.A new technique for producing of novel solvent-exfoliated graphene-TiOublished , which al method  was deve2-graphene oxide nanocomposite, which was prepared by thermal hydrolysis of suspension with graphene oxide nanosheets and titania peroxo-complex. It should be mentioned that we are able to produce pure graphene nanosheets in large quantity from natural graphite by using high intensity cavitation field in the high-pressure ultrasonic reactor. Graphene oxide sheets, prepared by such safe and friendly method, can be used as a good support for TiO2 to enhance its photocatalytic activity. In this paper, we reported on an "one-pot" thermal hydrolysis of titanium peroxo-complexes in the presence of graphene oxide in aqueous solution. Our "hydrogen peroxide route" is based on hydrolysis of Ti(IV) sulfate, next treatment of the resulting gel by hydrogen peroxide, and thermal decomposition of so formed peroxo-titanate under reflux at ambient pressure producing directly the TiO2-GO photocatalyst. The relative photocatalytic activity of the as-prepared thin layers of titania/graphene nanocomposite in poly(hydroxyethyl methacrylate) was assessed by the photocatalytic decomposition of butane under UV and visible light.In our previous work , we repo4, hydrogen peroxide H2O2, ethylene glycol OHCH2CH2OH and ammonium hydroxide NH4OH were supplied by Sigma-Aldrich. Graphene was produced in large quantity from natural graphite (Koh-i-noor Grafite Ltd. Czech republic) using high intensity cavitation field in a pressure batch-ultrasonic reactor  [All chemical reagents used in the present experiments were obtained from commercial sources and used without further purification. Titanium oxo-sulfate TiOSOcs GmbH) .2SO4 and 10 ml of H3PO4, 1 g of graphene and 3 g of KMnO4 were mixed in round bottom flask. The reaction was then heated to 40°C and stirred for 6 hours and pink squash suspension was obtained. Subsequently was poured onto ice with 200 ml of 30% H2O2. The pink squash suspension quickly changed to lemon-like yellow suspension. The whole reaction product was purged by dialysis , washed with ethanol and dried at 105°C.Graphene oxide was prepared by our safety method, a 60 ml of H2 - GO nanocomposite preparation, 100 ml of 1.6 M titanium oxo-sulfate (TiOSO4) was hydrolyzed by slow addition of ammonium hydroxide solution (10%) under constant stirring at temperature of 0°C in ice bath. The stirring last until the reaction mixture reaches pH 8.0. The obtained white precipitate was separated by filtration. The consequent depuration of sulfate ions from precipitate with distilled water was confirmed by the BaCl2. The wet precipitate is mixed with 100 ml of 15% hydrogen peroxide solution; thereby a yellow solution of titania peroxo-complex is obtained.In the typical procedure of TiO2 - GO nanocomposite was filtered off and dried at 105°C. Ten samples of TiO2-GO nanocomposites doped as TiGO_XXX, where XXX are grams of GO, were prepared.Well defined quantity (see Table 2 - GO nanocomposite powder (2 g) was dispersed in a mixture of 5 ml poly(hydroxyethyl methacrylate) and 10 ml of ethanol. From this suspension, a 300 μm thin layer on a piece of glass, 100 × 150 mm was created.The prepared TiODiffraction patterns were collected with diffractometer PANalytical X´Pert PRO equipped with conventional X-ray tube  and a linear position sensitive detector PIXcel with an anti-scatter shield. A programmable divergence slit set to a fixed value of 0.5 deg, Soller slits of 0.02 rad and mask of 15 mm were used in the primary beam. A programmable anti-scatter slit set to fixed value of 0.5 deg., Soller slit of 0.02 rad and Ni beta-filter were used in the diffracted beam. Qualitative analysis was performed with the DiffracPlus Eva software package  using the JCPDS PDF-2 database . For quaAFM images were obtained using an NTEGRA Aura (NT-MTD) microscope. A sample of the diluted dispersion was placed on synthetic mica as an atomically smooth support and evaporated at room temperature. The measurements were performed in air at room temperature in non contact mode, with Si tips of the 1650–00 type at resonance frequencies ranging from 180 to 240 kHz.The morphology of sample powders was inspected by transmission electron microscopy (TEM) and the crystal structure was analyzed by electron diffraction (ED) using a 200 kV TEM microscope JEOL 2010 F. As specimen support for TEM investigations a microscopic copper grid covered by a thin transparent carbon film was used. The samples were studied in both bright field and by electron diffraction with a selecting aperture (SAED) mode at an acceleration voltage of 200 kV.The surface areas of samples were determined from nitrogen adsorption–desorption isotherms at liquid nitrogen temperature using a Coulter SA3100 instrument with outgas 15 min at 150°C. The Brunauer–Emmett–Teller (BET) method was used for surface area calculation , the porThe Raman spectra were acquired with DXR Raman microscope (Thermo Scientific) with 532 nm (6 mW) laser, 32 two-second scans were accumulated with laser 532 nm (6 mW) under 10× objective of Olympus microscope.-1, respectively, with single-reflection horizontal accessory on Si crystal. The samples were mixed with KBr and pressed to conventional pellets at ambient conditions and measured in the transmission mode.Infrared spectra were recorded by using Thermo-Nicolet Nexus 670 FT-IR spectrometer approximately in 4000–500 and 500–50 cm-8 mbar. XPS survey-scan spectra were made at pass energy of 40 eV; the energy resolution was set to 0.5 eV. While individual high-resolution spectra were taken at pass energy of 10 eV with 0.05 eV energy steps. A software tool CasaXPS was used to fit high-resolution multi components peaks. The proper surface charge compensation was done by fitting C-C, C-H component of C 1 s peak to reference binding energy 284.5 eV. The atomic concentration of compounds was evaluated with relative sensitivity factors (RSF) defined in standard table of CasaXPS software.XPS apparatus was equipped with SPECS X-Ray XR50 (Al cathode 1486.6 eV) and SPECS PHOIBOS 100 Hemispheric Analyzer with 5-channels detector. A background pressure in XPS during the measurements was under 2×104 as a standard was used. The reflectance data were obtained as relative percentage reflectance to a non absorbing material (BaSO4) which can optically diffuse light.Diffuse reflectance UV/VIS spectra for evaluation of photo-physical properties were recorded in the diffuse reflectance mode (R) and transformed to absorption spectra through the Kubelka-Munk function . A Perki-2). The gas concentration was measured with the use of Quadrupole Mass Spectrometer JEOL JMS-Q100GC and gas chromatograph Agilent 6890 N. A high-resolution gas chromatography column  was used. Samples were taken from the reactor automatically through the sampling valve  in a time interval of 2 hours.Kinetics of the photocatalytic degradation of butane (0.87%) was measured by using a home-made stainless steel batch photo-reactor  with a N2, and consequently neither butane absorbed on the poly(hydroxyethyl methacrylate) matrix. The injection volume of butane into the photoreactor was 30 ml.Blank tests (a layer of poly(hydroxyethyl methacrylate) without titania) were performed in order to establish the effect of photolysis and catalysis on the conversion of butane. The UV irradiation detects that there was none or immeasurable conversion of butane, as a testing gas, into CO and/or CO001 = 6.33 Å [001 = 6.718 Å which is in good agreement with published results [Early published experimental XRD pattern of graphene oxide demonstrated arising of strong 001 reflection peak at 2θ ~10° with a basal spacing of d= 6.33 Å . We calc results ,17.2-GO nanocomposites. All samples show only the anatase phase (PDF 21–1272). Higher concentrations of GO (samples TiGO_300 and TiGO_400) probably lead to partial reduction of GO to graphene and a weak peak at 2Θ = 26.50° appears (see arrow G). Nevertheless, the intensity of graphene X-ray lines is under possibility for quality Rietveld refinement. Furthermore, in the position at 2Θ ~ 10-12° (see arrow GO), there is an obvious hint of GO peak, which is due to the small particle size and its concentration featureless.The Figure 4+ are transformed to Ti3+, because of the larger ionic radius of Ti3+ (0.670 Å) compared to Ti4+ (0.605 Å) [Crystallite size, a and c cell parameters of anatase, calculated by the Rietveld refinement procedure using Topas v.4.2 programme, based on the full width at half-maximum of the peak at 25.4° are presented in Table 0.605 Å) .2-GO nanocomposite.Atomic force microscopy and electron transmission microscopy were used to determine quality of delamination of graphene oxide (GO) used for preparation TiO2 nanoparticles and graphene oxide sheet prevents the re-aggregation of the sheets of graphene oxide. The TEM images of GO prepared by modified oxidation method is presented in Figure 2-GO nanocomposite and demonstrates that TiO2 nanoparticles were dispersed uniformly on the graphene plane.In our previously work we have been demonstrated that the thermal hydrolysis of titania peroxo-complex leads to spindle-like particles . Our expFigure 2-GO nanocomposites calculated by the multi-point Brunauer-Emmett-Teller (BET) method and total pore volume are listed in Table 2-GO nanocomposites have mesoporous texture. With increasing content of GO in the composite decreasing trend of specific surface area is obvious  was found compared to the theoretical value of the graphene and graphene oxide, respectively. Nevertheless mesoporous TiO2-graphene oxide nanocomposites have specific surface area in interval ~ 80–200 m2g-1 being considerably larger than those of P25 and similarly prepared neat TiO2 particles without using graphene oxide.The specific surface area of the prepared TiO4+ to Ti3+[4+, somewhere an oxidation must occur, and it is probably on the graphene skeleton in free positions of Π bonds. Following oxidation of graphene due to reduction of Ti4+ corresponds to an increase of intensity of Raman bands GO in the nano-composite TiO2-GO.Raman spectroscopy is a powerful tool to characterize the crystalline quality of graphene or GO. Additional file + to Ti3+. However-1, whereas the intensity of the D band at 1350 cm-1 increases substantially. Increased background of the spectrum is caused by the sample fluorescence. G band is common to all sp2 carbon forms and provides information on the in-plane vibration of sp2 bonded carbon atoms and the D band suggests the presence of sp3 defects [In our Raman spectrum, the G band is broadened and shifted slightly to 1605 cm2-GO is presented in Figure -1 (Eg), 396 cm-1 (B1g), 512 cm-1 (B1g + A1g) and 631 cm-1 (Eg) indicating the presence of the anatase phase in all of these samples. The D bands of GO are located at 1349 cm-1 and G band at 1601 cm-1. With increasing concentrations of GO the intensity of both bands also increases. The background of the sample labeled TiGO_075 is increased by the sample fluorescence.The Raman spectra of series nanocomposites TiO2-GO nanocomposites was evaluated from the high resolution spectra of O 1 s, C 1 s and Ti 2p. The compositions are summarized in the Additional file High resolution XPS spectra were accumulated 10 times to enhance the signal and to noise ratio, and are presented in Figure 4)2 with GO, plenty of oxygen-containing groups, such as C = O located in the interlayer of graphene oxide were consumed during the nucleation and growth of TiO2 crystallites, which means graphene oxide has been partly reduced [2 nanoparticles, resulting in hydrogen bonds between them [The binding energy calibration was made on C-C component of C 1 s peak after a deconvolution process. The intensity of C 1 s peak is rather small and the signal is noisy, but the C 1 s peak was fitted by two main components to represent C-C/C-H bond at binding energies about 284.5 eV and C = O bond at higher binding energies about 288 eV. The C = O bond was expected because it is a part of the graphene oxide precursor. Furthermore,during reaction of Ti and had C-C/C-H character; therefore it can be expected that the carbon comes from the precursor rather then from additional contaminations. The other expected bonds as O = C-OH and C-OH were neglected because of the weak signal .3/2 and Ti 2p1/2 were identified at binding energies of 458.5 and 464.1 eV, respectively. The position of Ti 2p peaks corresponds to the literature reported values and also to the FWHM values about 1.6 eV for Ti 2p3/2 and 2.4 eV for Ti 2p1/2, which are reasonable for single peak character. The Ti is probably bonded in the TiO2. The equal area/RFS ratios were found on both spin states within precision about 10%. The limited precision is due to the background removal process when the Shirley background was used. The peak positions and the FWHM were found to be practically identical for all investigated samples.The Ti 2p2-GO nanocomposite prepared by thermal hydrolysis of titania peroxo-complexes. The broad absorption peaks about 3427 cm-1, and the band at 1631 cm-1 correspond to the surface absorbed water and the hydroxyl groups [-1 can be assigned to hydroxyl C-OH and that at 1258 cm-1 to epoxide C-O-C [-1 is most probably related to the Ti-O vibration modes of the Ti3+-O-Ti4+ framework [The Additional file l groups . The bande C-O-C . The ban2-GO nanocomposite. The presence of different amounts of GO influences the optical properties of light absorption significantly. With increasing GO content in the nanocomposite light absorption intensity in the UV region grows and a red shift to higher wavelength in the absorption edge at about 400 nm also observed [2[3+.The Additional file observed . Increasserved [2 and part2/2R, R is the reflectance of the "infinitely thick" layer of the solid [1/2 versus photon energy (hν = 1239/λ) [2 is 3.20 eV, [2-GO nanocomposite has been reduced from value 3.15 eV (sample TiGO_001) to value <2.50 eV for sample TiGO_100. The band-gaps of samples TiGO_200 to TiGO_500 are impossible to be identified because the absorption edges for TiO2 entirely overlapped with those for GO.According to the equation for band gap:he solid , B is ab 1239/λ) ,31, whic3.20 eV,  whereas The effect of oxidation time variation on the electronic, physical and chemical properties of GO was investigated and it was found that the bandgap of GO increased with oxygen content of the GO material . GO has 2 to form a Ti–O–C structure, which then shifted up the valence band edge and reduce d the band gap [Graphene oxide is non-stoichiometric material, which retains the lamellar structure of graphite. After treatment in the presence of strong acids and oxidation agents, many oxygen-containing groups such as carboxyl (C − OOH), hydroxyl (C − OH), epoxide (C − O − C) become covalently attached to its layers surfaces. In our experiment GO reacted in the presence of Ti-peroxo-complex, and some functional group such as  on the GO surface were removed. We can can suppose that some unpaired ∏ − electrons bonded with the free electrons on the surface TiOband gap .- and h+ is strongly reduced, which increases the process yield.On another hand after the photoactivation of titanium dioxide the electrons can easily transfer to the graphene-nanosheets and photoinduced holes migrate into titania; recombination of e2-GO nanocomposites were assessed from the kinetics of the photocatalytic degradation of butane in the oxygen atmosphere. Photocatalytic oxidation of butane are based on the following overall reactions [The photocatalytic activity of the as-prepared layered of TiOeactions :(2)2C4H12 and the measurement is run. If the measured gas chromatogram shows only the oxygen peak at retention time 1.52 min then a gas for the photocatalytic decomposition (in our case the butane) can be injected. After measuring the subsequent chromatogram, peaks for the oxygen and the butane are marked each by value of 1. The value of gases, which are expected to be formed due to photo-activity of the TiO2-GO nanocomposite  are assigned each by value 0. By this normalization of input data is performed and the measurement with time period of 2 hours can be started.Before each measurement, the photocatalytic reactor is evacuated, then rinsed with the O2 and H2O after time reaction 60 hours is presented in Table In a typical chromatograph of the effluent obtained from butane photocatalytic degradation only oxygen, carbon monoxide, carbon dioxide, water and butane were detected at retention times of 1.52, 1.54, 2.41, 6.42 and 9.53 min . The nor0 is the initial concentration, c is the concentration of butane after time (t), and k is the first-order rate constant.The typic corresponding experimental dependencies of butane, oxygen, carbon monoxide and carbon dioxide in time are plotted in Figure 2 at ambient temperature. In general, they observed that alkanes (CnH2n+2) formed ketones (CnH2nO) and other aldehydes CmH2mO with 2 < m < n. If the alkane was branched, the ketone was CmH2mO with 3 < m < n. The reactivity of different types of carbon atoms followed the sequence: Ctertiary > Cquaternary > Csecondary > Cprimary. Paper [2 and H2O.The photocatalytic degradation of butane was fitted by curves of the first-order kinetics; the corresponding rate constants are given in Table y. Paper  shows, t-1) and TiGO_075 (k = 0.00774 h-1), respectively. As for the TiGO_075 sample, there is also the largest increase in CO and CO2 content. It is evident from Table 2 at the expense of the primary decomposition of butane and the value for rate constant decreases. This suggests that in these samples a much deeper mineralization occurs, which leads to the preferential decomposition of intermediates to end products of photocatalytic decomposition, i.e. CO2 and H2O. This increase in photoactivity for some samples is probably due to reduction of Ti4+ to Ti3+ and oxidation of free ∏ − bonds during preparation. The interaction of CO2 molecules with the excited states of Ti3+-O- leads to the formation of CO2•¯ anion radicals [2•¯ anion radicals readily react with poly(hydroxyethyl metacrylate), which leads to the formation of carbon monoxide due to its strong adsorption on Ti sites of the TiO2 surface [2 nanoparticles, ii) graphene oxide sheets can enhance adsorption ability of the TiO2 - graphene oxide nanocomposite, iii) Graphene oxide works as electron acceptor and photosensitizer to efficiently enhance the butane photodecomposition.The results presented in Table radicals . The CO2 surface . The fun2, we developed a simple ("one-pot") method to prepare nanocomposite by depositing TiO2 on the graphene oxide through liquid phase deposition.Using graphene oxide sheets as a substrate for TiO2-GO nanocomposite systems irrespective of light sources could be attributed to synergy effects including the increase in specific surface area with graphene oxide amount as well as to the formation of both π–π conjugations between butane molecules and aromatic rings and the ionic interactions between butane and oxygen-containing functional groups at the edges or on the surfaces of carbon-based nanosheets. Graphene oxide works as the adsorbent, electron acceptor and photosensitizer and efficiently enhances the butane photodecomposition. The best photocatalytic activity under UV and visible light was observed for samples denoted TiGO_100 (k = 0.03012 h-1) and TiGO_075 (k = 0.00774 h-1), respectively. Both the used synthesis route and the GO modification are promising ways to cheap and efficient photocatalysts for the Vis light activation.The better photocatalytic properties on the TiOThe authors declare that they have no competing interests.VŠ was the main author of the work, performed syntheses, and coordinated all characterization and catalytic studies. SB was responsible for electron microscopy, TMG for Raman spectroscopy, MK for XPS measurement, JB assisted with manuscript writing. All authors read and approved the final manuscript.Figure S2. IR spectrum of the TiO2-GO nanocomposite. Figure S3. UV–vis absorption spectra of the TiO2-GO. Table S1. C Composition of nanocompsite base on XPS.The Raman spectrum of prepared graphene oxide. Click here for file


D/IG down to < 0.02 in Si-face samples and < 0.05 in C-faces over the 1 cm2 wafer surface grown at 1,450°C. AFM examination shows a better morphology in face-down surfaces. This study suggests that the optimum configuration slows the thermal decomposition and allows the graphene to form near the equilibrium. The Si-face-down orientation (in opposition to the temperature gradient) results in a better combination of low disorder ratio, ID/IG, and smooth surface morphology. Mobility of Si-face-down orientation has been measured as high as approximately 1,500 cm2/Vs at room temperature. Additionally, the field effect transistors have been fabricated on both Si-face-down and C-face-down showing an ambipolar behavior with more favorable electron conduction.We have explored the properties of SiC-based epitaxial graphene grown in a cold wall UHV chamber. The effects of the SiC surface orientation and silicon loss rate were investigated by comparing the characteristics of each formed graphene. Graphene was grown by thermal decomposition on both the silicon (0001) and carbon (000-1) faces of on-axis semi-insulating 6H-SiC with a "face-down" and "face-up" orientations. The thermal gradient, in relation to the silicon flux from the surface, was towards the surface and away from the surface, respectively, in the two configurations. Raman results indicate the disorder characteristics represented by I Then, devices were fabricated on these graphene films. Figure -1 and 2,710 cm-1, respectively. The Si-face-down surface showed the narrowest G-peak with the full width at half maximum of 19 cm-1. Defect related D-peaks were also found at around 1,350 cm-1, and the relative intensity of D-peak and G-peak in the Raman spectra was used to estimate the quality of graphene and the degree of disorders. This ID/IG ratio has been known to be inversely proportional to the grain size and provides a good estimation for the quality of graphitic materials  with a magnetic field of 0.2 T. RT mobility as high as approximately 1,500 cm2/Vs was measured from the graphene on Si-face-down sample with a sheet carrier density of 2 to approximately 3 × 1013 cm-2. As reported previously on epitaxial graphene, mobility increased as carrier density decreased [2/Vs. The degradation of the field effect mobility is likely caused by the dielectric layer on top of the graphene as well as the effects of device fabrication.The signature of graphene as measured by Raman spectroscopy was observed in all the samples. Figure aterials . The ID/ecreased . Figure ecreased . Specialecreased ) or fixeD/IG ratio below 0.02 with smooth and uniform surface morphology. Electrical properties were characterized by test structure-fabricated and Hall-effect measurements. In transistor characteristics, it shows an ambipolar behavior, and the electron conduction is favored over the hole conduction, which is determined by the polarity of initial majority carriers in graphene layer during the formation of graphene. The Hall mobility at RT is as high as 1,500 cm2/Vs, and the value of field effect mobility is close to that of silicon devices. This approach to form a graphene film could be used to grow high quality epitaxial graphene in case a relatively lower-annealing temperature is required.By comparing the face-down (low Si evaporation rate) and face-up (high Si evaporation rate) mounting method, the optimum growth condition producing a higher quality graphene in a cold wall UHV chamber was found. The use of graphite enclosure and the face-down scheme provide a condition that can reduce Si loss rate and allow higher temperature growth. The best quality film was obtained with the Si-face-down configuration with the IThe authors declare that they have no competing interests.MK and JH contributed equally to this work. They carried out the graphene growth and device fabrication/characterizations. VS participated in the growth of graphene and material characterizations. ST participated in the design of devices and its coordination. MS and J-WL conceived of the study and design most of this project. All authors read and approved the final manuscript.


Foliar fungal communities of plants are diverse and ubiquitous. In grasses endophytes may increase host fitness; in trees, their ecological roles are poorly understood. We investigated whether the genotype of the host tree influences community structure of foliar fungi. We sampled leaves from genotyped balsam poplars from across the species' range, and applied 454 amplicon sequencing to characterize foliar fungal communities. At the time of the sampling the poplars had been growing in a common garden for two years. We found diverse fungal communities associated with the poplar leaves. Linear discriminant analysis and generalized linear models showed that host genotypes had a structuring effect on the composition of foliar fungal communities. The observed patterns may be explained by a filtering mechanism which allows the trees to selectively recruit fungal strains from the environment. Alternatively, host genotype-specific fungal communities may be present in the tree systemically, and persist in the host even after two clonal reproductions. Both scenarios are consistent with host tree adaptation to specific foliar fungal communities and suggest that there is a functional basis for the strong biotic interaction. Endophytic fungi live in the tissues of leaves and other plant organs without causing symptoms of disease in planta diversity; class 3: broad host range, mostly above-ground tissues of plants, horizontal infection of hosts, high in planta diversity; class 4: broad host range, present in roots). Vertically transmitted endophytes typically infect their hosts during reproduction, while horizontally transmitted endophytes randomly infect their hosts from environmental sources. The group typically associated with tree leaves is the "class 3", nonclavicipitaceous endophytes. Class 3 endophytes are highly diverse, occur in the above-ground tissues, and are generally considered to be horizontally and stochastically distributed Host plant characteristics are known to influence fungal community assembly. It has been shown that plant genotype, taxonomic identity, as well as specific plant traits such as chemical properties can affect microbial community composition and diversity Populus balsamifera L.). This is a well-studied North-American tree species, with a vast range covering most of Canada, Alaska, and the northern U.S.A. The current distribution is the result of a northward range expansion after the last glacial maximum In this study we investigate potential effects of the host genotype in shaping the composition of the foliar fungal communities of balsam poplar  since 2007. In 2009 cuttings from these trees were sent to Fairbanks, Alaska, rooted in the greenhouse, and planted into a new common garden. The newly rooted trees originating from these cuttings grew for 12 months in the Fairbanks common garden until our sampling in 2010. The Fairbanks garden is located on a cleared area of the University of Alaska campus . The opening is surrounded by coniferous forest. All trees were genotyped using single nucleotide polymorphisms (SNP) from 590 gene fragments To ensure that the fungal communities did not change during transportation to the lab we rapidly dried the leaves by placing them immediately in silica gel. Leaves dry very rapidly under these conditions (within a few hours from the time of the sampling). We assumed that the rapid drying also prevents preferential fungal growth in the collected leaves . Once dried, the sampled leaves were carried to the lab in plastic bags filled with silica gel. Within 2 months of sampling, DNA was extracted from dried leaves using Qiagen DNeasy Plant Mini Kit (Qiagen Inc. USA), without surface-sterilization. We are aware that avoiding surface-sterilization may cause further complexity in the data. We consider that our dataset represents both endophytic communities and fungi found on the surfaces of the leaves.DNA extraction, PCR conditions, and primers can strongly influence community composition recovered by amplicon sequencing CTTGGTCATTTAGAGGAAGTAA) and ITS4 (TCCTCCGCTTATTGATATGC) fungal primers Using ITS1F . We quantified the tagged PCR product concentration with Quant-iT™ PicoGreen®dsDNA assays . MID-tagged community amplicons from five additional, non-genotyped poplar specimens were also multiplexed in the same sequencing run, along with the PCR products for the current study s = 60, PyroNoise initial clustering cutoff c = 0.01; SeqNoise cluster size s = 30, SeqNoise initial clustering cutoff c = 0.08). Trimmed reads shorter than 300 bp were discarded, and all reads were truncated to 450 bp to remove noisy ends. Chimera checking was performed with PerseusD After removing MID tags, binary SFF files were converted into text flowgrams with sffinfo v2.5.3, a program from the 454 Sequencing System software (454 Life Sciences). Trimming information provided by the sequencer in the raw data files was used for a preliminary trimming of the 3′ ends of poor quality fragments of the flowgrams with sffinfo. The trimmed flowgrams were processed with the AmpliconNoise v1.23 pipeline We downloaded all annotated fungal ITS1, 5.8S, and ITS2 sequences from NCBI  and used these to build a BLAST v2.2.21 We removed all sequences not identified as being of fungal origin by the previous BLAST/MEGAN assignment. For the rarefaction analysis we clustered the remaining forward reads  with a grammar-based approach in GramCluster v1.3 We analyzed fungal community structures by selecting all BLAST hits assignable to the lowest taxonomic level . We completed this matrix with all hits assigned to the genus level. This resulted in a taxonomic matrix consisting almost exclusively of species and genera, containing also six families . We omitWe used linear discriminant analysis (LDA) as implemented in the R package MASS The 454 Titanium runs on two 1/4th plate, and two 1/16th plate fractions produced 204,052 reads. Of these reads 203,459 featured correct MID tags (corresponding to the 28 samples multiplexed for sequencing). After quality trimming of read ends, filtering for overall sequence quality and chimeras, and discarding short (less than 300 bp) reads, 126,402 reads were retained . The final number of reads after filtering for 5′-3′ oriented forward sequences was 51,596. Individual samples contained 1,388–3,057 forward-oriented reads.The combined BLAST/MEGAN analysis assigned the reads to 125 taxa. The communities were dominated by a few very abundant taxa. However, most of the taxa were rare, 80% of them being represented by less than 43 reads combined in the 23 host clone leaves . Individgof = 0.475).We plotted different similarity thresholds and showed that sequence cluster discovery approaches, but does not reach, a saturation plateau . The spea priori groups defined on the basis of their host genotypes .Linear discriminant analyses revealed that the genotype of the poplar trees and the composition of their foliar fungal community are tightly linked . Most foenotypes . Of the enotypes . The strOur survey of the leaf-associated fungal communities found in and on poplar leaves sampled from a common garden revealed that the host genotype is a significant determinant of the composition of leaf-associated fungal communities. This finding is in conflict with the expectation that foliar fungal communities represent a random sample of fungi present in the environment. The results imply the existence of specific relationships between the host tree and its foliar fungal microbiome. Intriguingly, the geographic divergence of the balsam poplar subpopulations happened relatively recently, after the last glacial maximum , relatives of known plant parasites (Mycosphaerella sp.). Some reads were assigned to unexpected taxa, e.g. Blumeria graminis and Pyrenophora tritici-repentis (both known to occur only in Poaceae), Leucosporidium golubevii (known from freshwaters). The unexpected assignments may also result from epiphyllous diaspores. Alternatively, the unexpected hits may come from presently unknown taxa, which are happen to be closely related to species present in GenBank. The list of BLAST hits should also be interpreted cautiously, as it strongly relies on the quality of our reference database  are known as ballistosporic yeasts without host specificity. The genus Fusicladium  includes almost 100 species, some of them known to be highly host specific, as shown for example on Populus spp. by Newcombe sensu Pompanon et al. Each of the 25 most common taxa had some discriminating value in the LDA, but only two of them had above-average discriminating values . SporoboSimilar to previous reports on tree leaf endophytes characterized by 454 pyrosequencing Two basic mechanisms possibly explain the patterns observed in the present study. First, host traits may promote infections with specific strains from the environment. Host morphology, physiology Our results show that foliar fungal communities of trees growing in a common garden show host genotype-specific structures. This raises the possibility that at least some members of the leaf fungal community adapt to plant genotypes. Given that many endophytes alter plant fitness, this could be a form of coevolution. We hypothesize that host-foliar fungal relationships in trees might have been obscured by the low resolution of cloning and culturing methods available before next-generation metabarcoding. Most of the strongly discriminating taxa were relatively rare in the foliar fungal communities sequenced in this study . TechnolTable S1a priori LDA groups and LDA results of foliar fungal communities from balsam poplar.Collection data, 454 MID tags of the samples during sequencing, (XLS)Click here for additional data file.Table S2List and functions of foliar fungal taxa discriminating among host balsam poplar specimens on the basis of genotype group and regional translocation events.(XLS)Click here for additional data file.Material S1Pruned sequences produced by the 454 runs for the foliar fungal communities of each balsam poplar host specimen in FASTA format.(ZIP)Click here for additional data file.Material S2The number of pruned 454 reads assignable by MEGAN to low-level taxa (125), on the basis of BLAST hits against annotated fungal sequences present in GenBank.(ZIP)Click here for additional data file.Material S3Clusters delimited at sequence-divergence based equivalents of 6 grammar thresholds with GramCluster.(ZIP)Click here for additional data file.


More specifically, we quantify the effect of either pharmacological upregulation of the heat shock response or specific genetic upregulation of a small heat shock protein, CG14207, on the neurotoxicity of both TDP-43 and of its disease associated 25 kDa fragment (TDP-25) in a Drosophila model. Inhibition of the aggregation of TDP-43 by either method results in a partial reduction of its neurotoxic effects on both photoreceptor and motor neurons, whereas inhibition of the aggregation of TDP-25 results not only in a complete suppression of its toxicity but also its clearance from the brain in both neuronal subtypes studied. The results demonstrate, therefore, that aggregation plays a crucial role in mediating the neurotoxic effects of both full length and truncated TDP-43, and furthermore reveal that the in vivo propensity of these two proteins to aggregate and their susceptibility to molecular chaperone mediated clearance are quite distinct.Almost all cases of sporadic amyotrophic lateral sclerosis (ALS), and some cases of the familial form, are characterised by the deposition of TDP-43, a member of a family of heteronuclear ribonucleoproteins (hnRNP). Although protein misfolding and deposition is thought to be a causative feature of many of the most prevalent neurodegenerative diseases, a link between TDP-43 aggregation and the dysfunction of motor neurons has yet to be established, despite many correlative neuropathological studies. We have investigated this relationship in the present study by probing the effect of altering TDP-43 aggregation behaviour  Although the ability to form intractable aggregates is an intrinsic property of all polypeptide chains, the formation of such species under physiological conditions is largely restricted to a subset of proteins associated with progressive pathological conditions In order to investigate the molecular origins of ALS and related disorders, a number of cellular and animal models of TDP-43 expression have now been established. Whilst some of these models have indicated a toxic gain of function role for TDP-43 in protein aggregation and neurodegeneration, others have suggested that the cause of neuronal dysfunction is the loss of normal TDP-43 function, or indeed that the retention of the RNA binding activity of TDP-43 resulting in aberrant binding is a cause of its toxicity in vivo, clearly indicate that these molecules play an important role in the regulation of pathological protein aggregation In the present study we set out to investigate specifically the role of aggregation of full length TDP-43 and of an N-terminally truncated and disease-associated fragment, TDP-25, in causing neurodegeneration by upregulation of molecular chaperones, the inherent machinery of all biological systems that combats misfolding and aggregation; it is well established that molecular chaperones are able to inhibit the aggregation of proteins associated with neurodegenerative diseases Drosophila melanogaster, and these model systems have been formed to capture many of the key molecular features of these diseases Drosophila model of TDP-43 proteinopathy and demonstrate that both pharmacological and genetic upregulation of chaperones enable a dissection of the distinct roles played by aggregation in neurodegeneration induced both by TDP-43 and its N-terminally truncated fragment TDP-25.Many neurodegenerative disorders, including AD, PD and Huntington's disease (HD) have been modeled in Drosophila were generated, each expressing one of these proteins. In the first line, the human TARDBP gene was introduced into the Drosophila genome using a modified UAS-GAL4 vector Drosophila genome in the same way , two lines of transgenic same way  [17]. BoDrosophila typically cause a characteristic ‘rough eye’ phenotype in which the normal linear array of ommatidia and sensory bristles become disordered, and in addition the eye loses its natural pigmentation to varying degrees that depend on the toxicity of the proteins expressed gmr-GAL4 driver was found to result in a rough eye phenotype in both cases, although its severity was observed to vary dramatically between the two genotypes. Thus, flies expressing TDP-43 have a strong rough eye phenotype, characterised by an almost complete absence of structured ommatidia when examined by either optical or scanning electron microscopy, whereas TDP-25 flies show only a weak phenotype that is only clearly evident on high magnification analysis using scanning electron microscopy , a disease that exhibits a similar cytoplasmic aggregation phenotype to the TDP-43 proteinopathies Drosophila, we decided that CG14207 was an excellent candidate to establish whether chaperones can affect the aggregation and the subsequent toxicity of TDP-43 and TDP-25.17-AAG is known to have pleiotropic effects on the regulation of multiple cellular processes, including autophagy and apoptosis, as well as inducing chaperone expression Flies overexpressing CG14207 were crossed with those expressing TDP-43 and TDP-25, and the effects on their neurotoxic phenotypes were monitored. A very similar pattern of results was obtained to those observed with 17-AAG treatment, as CG14207 expression is only partially able to rescue the neurotoxic effect of TDP-43 on developing photoreceptors , and redgmr-GAL4 driver was used, which was able to drive expression of TDP-25 to a much higher level than that achieved by the driver used in In a similar series of experiments with flies expressing TDP-25, where the TDP-25 neurotoxic rough eye phenotype is mild in comparison to that elicited by TDP-43 expression, complete rescue upon CG14207 co-expression was observed. It was important to determine whether the difference in the extent of the rescue by CG14207 of TDP-43 and TDP-25 toxicity was indeed due to the two proteins having different intrinsic susceptibilities to CG14207 activity or is attributable to the apparently lower steady state concentration of TDP-25 present compared to TDP-43. To address this question a stronger D42-GAL4 driver). The median lifespans of the flies in which TDP-43 or TDP-25 expression was driven in these motor neurons were reduced by 57±2% and 17.7±3%, respectively, compared to control flies , such as α-crystallin and αB-crystallin may prevent the formation of insoluble, amyloid fibrils of other proteins but, depending on their stoichiometric ratios with respect to their client proteins, may also stabilise or even enhance the formation of relatively soluble but potentially neurotoxic non-fibrillar aggregates We also find that chaperone upregulation, and subsequent reductions in the microscopic and biochemical detection of aggregates of TDP-43, is associated with a reduction in its pathogenic effects demonstrated by photoreceptor degeneration or motor neuron dysfunction. In the case of flies expressing TDP-43, unlike those expressing TDP-25, this reduction in toxicity is incomplete, a finding consistent with the continued presence of aggregated TDP-43 that we have demonstrated biochemically by the detection of urea insoluble protein, albeit at much lower levels, in the brains of in vivo aggregation behaviour of TDP-43 and TDP-25, and its relationship with neurotoxicity. Our results demonstrate that whilst both TDP-43 and TDP-25 form aggregates in vivo, the abundance of these aggregates, the ease with which they can be prevented from forming by the small heat shock protein CG14207 and their neurotoxic effects are very substantially different. The important role of aggregation in the TDP-43 proteinopathies is therefore consistent with our understanding of other protein misfolding diseases such as AD, PD and HD.In conclusion we have exploited the upregulation of chaperones to probe the D. melanogaster and synthesised by Genscript . Each construct was cloned into the multiple cloning site of pUAST-AttB and then subcloned into a modified pCasper vector . Differences in the expression of the constructs that could arise from their integration at different genomic loci were therefore eliminated as the pCa4B vector contains sites for exploiting the PhiC31 system for site-specific integration of transgenes. All injected constructs were incorporated at the same genomic locus (51D) on Chr. II  HA-tagged TDP-43 constructs were codon optimised for expression in gmr-GAL4 flies (Bloomington Stock ID: 1104 & 8121). All flies were kept in a temperature and humidity controlled incubator maintained at 25°C, 70% humidity. Optical microscope pictures were taken of 1-day-old TDP/gmr-GAL4 offspring at a magnification of 7.5×. SEM images were collected as described previously TDP-43 and TDP-25 flies were crossed with Third-instar larval imaginal eye discs and adult brains were dissected, fixed in 4% PFA and blocked in 5% BSA in 0.05% PBS-Triton. This procedure was followed by incubation overnight with rat anti-HA-biotin high affinity antibody  and mouse anti-ubiquitin antibody . Discs or brains were then incubated overnight in Streptavidin Alexa Fluor 594 conjugate (Invitrogen) and anti-mouse FITC, and counterstained with TOTO-3 (Invitrogen) to detect nucleic acids.Five flies per genotype were decapitated, homogenised in RIPA buffer and centrifuged to pellet the insoluble forms of proteins. The pellets were then resuspended in 9 M urea and centrifuged again to collect urea soluble protein. Each of the protein samples was separated on 4–12% Bis-Tris gel (Invitrogen) under reducing or non-reducing conditions and transferred to PVDF membranes (Millipore). Blots were blocked with 5% non-fat milk and then incubated with primary antibodies: anti-HA-biotin, high affinity (3F10) antibody (Roche) followed by incubation with anti-rat secondary antibody conjugated to HRP. Bands were detected using a SuperSignal® West Femto Maximum Sensitivity Substrate kit (Thermo Scientific). Loading controls using a mouse anti-β-actin antibody (Abcam) and anti-mouse HRP (Dako) were performed on all western blots.gmr-GAL4 flies and maintained on either standard cornmeal-agar food treated either with distilled water or 1.5 µM 17-AAG to allow the larvae to ingest the drug. Microscopy, immunohistochemistry and western blot analyses were performed on drug treated and control offspring as described above.The geldanamycin analogue 17-allylamino geldanamycin  was resuspended in ethanol and diluted in distilled water to a concentration of 1.5 µM. 300 µL aliquots of drug were added to the surface of pre-made fly food and allowed to dry for 2 days. TDP-43 and TDP-25 flies were crossed with D42-GAL4 is activated by heat shock and so simply moving flies from 25°C to 29°C induces expression in motor neurons. Median survivals for each vial were calculated using Kaplan-Meier survival statistics (n = 100 per experiment) and differences between genotypes were analysed using a Mann-Whitney U test (n = 10 tubes of 10 flies each).Crosses were set up at 25°C. Non-virgin females were kept 10 per vial and moved to 29°C within 24 hours of eclosion and were transferred to fresh food and counted every 2–3 days. The experiments were performed in a temperature-controlled incubator at 29°C. Temperature sensitive Gal80 Motor function was assessed by a climbing assay. Flies expressing TDP-43 and TDP-25 in motor neurons from the day of hatching, with and without CG14207, were generated in separate experiments as described above. 10 flies were placed into a single tube and analysed every second day after the initial heat shock; this procedure was repeated three times on the same tube of flies for each day. A climbing index score was calculated as described previously Figure S1Light microscope images demonstrating the effects of TDP-43 when expression is driven by gmr-GAL4, in the presence and absence of a non-toxic control protein (UAS-GFP).(TIF)Click here for additional data file.


Idiopathic pulmonary fibrosis (IPF) is a chronic, fibrosing interstitial lung disease that primarily affects older adults. Median survival after diagnosis is 2–3 years. The clinical course of IPF may include periods of acute deterioration in respiratory function, which are termed acute exacerbations of IPF (AEx-IPF) when a cause cannot be identified. AEx-IPF may represent a sudden acceleration of the underlying disease process of IPF, or a biologically distinct pathological process that is clinically undiagnosed. An AEx-IPF can occur at any time during the course of IPF and may be the presenting manifestation. The incidence of AEx-IPF is hard to establish due to variation in the methodology used to assess AEx-IPF in different studies, but AEx-IPF are believed to occur in between 5 and 10% of patients with IPF every year. Risk factors for AEx-IPF are unclear, but there is evidence that poorer lung function increases the risk of an AEx-IPF and reduces the chances of a patient surviving an AEx-IPF. The presence of comorbidities such as gastroesophageal reflux disease (GERD) and pulmonary hypertension may also increase the risk of an AEx-IPF. AEx-IPF are associated with high morbidity and mortality. Patients who experience an AEx-IPF show a worsened prognosis and AEx-IPF are believed to reflect disease progression in IPF. Current treatments for AEx-IPF have only limited data to support their effectiveness. The latest international treatment guidelines state that supportive care remains the mainstay of treatment for AEx-IPF, but also give a weak recommendation for the treatment of the majority of patients with AEx-IPF with corticosteroids. There is emerging evidence from clinical trials of investigational therapies that chronic treatment of IPF may reduce the incidence of AEx-IPF. Additional clinical trials investigating this are underway. Idiopathic pulmonary fibrosis (IPF) is a chronic, fibrosing interstitial pneumonia of unknown cause that occurs primarily in older adults . In the A universally agreed definition of an AEx-IPF has not been established. Clinical studies have used a variety of criteria to define AEx-IPF, which differ in factors such as whether chest X-ray, or high-resolution computed tomography (HRCT) is required, how hypoxemia is defined, and the conditions that must be ruled out -14. VariIn 2007, a consensus definition for AEx-IPF was proposed by an expert committee sponsored by the IPF Clinical Research Network and the National Heart Lung and Blood Institute (NHLBI), in an attempt to standardize the diagnostic criteria used across studies . This coThe true incidence of AEx-IPF remains unclear. Reported incidences vary widely across studies, due to the lack of a consistent definition and differences in study methodology and patient selection ,21. The A variety of patterns of acute lung injury have been observed in AEx-IPF . The mosSeveral hypotheses for the etiology of AEx-IPF have been proposed. AEx-IPF may represent a sudden acceleration of the underlying disease process due to unknown acute injury to the lung, or a biologically distinct pathological process due to a clinically occult condition, such as infection or gastroesophageal reflux disease (GERD) . As AEx-Although research is ongoing into gene expression patterns and the identification of biomarkers, the molecular mechanisms underlying AEx-IPF remain poorly understood. Activation of the immune system, disordered coagulation/fibrinolysis, and oxidative stress may all contribute to the pathophysiology of AEx-IPF. Immune cells  ,28,29, iEpithelial cell damage in patients with IPF is demonstrated by over-expression of matrix metalloproteinase (MMP)-7 , MMP-9 , and KreAn AEx-IPF can occur at any time during the course of IPF and, for some patients, may be the presenting manifestation ,11,23,39A higher degree of dyspnea   or of fiGERD, a common comorbidity in patients with IPF, may contribute to the pathogenesis of IPF through introduction of gastric acid into the respiratory tree ,47,48. I2, DLCO) prior to AEx-IPF are more likely to survive an AEx-IPF  vs. 7 [7.6%]), but the number of events was small and the difference was not statistically significant . In triaPirfenidone, an anti-fibrotic molecule that has been licensed for the treatment of IPF in Japan, India, China, Europe, and Canada, but was not approved in the United States, has shown inconsistent effects on AEx-IPF. A Phase II study in Japanese patients with IPF was terminated after 9 months of a planned 1-year follow-up based on a higher frequency of AEx-IPF in the placebo group than in the pirfenidone 1800 mg/day group . HoweverNintedanib (formerly known as BIBF 1120) is a tyrosine kinase inhibitor in clinical development for the treatment of IPF. In the Phase II, 12-month, randomized, placebo-controlled TOMORROW trial, a lower incidence of AEx-IPF was observed in patients treated with nintedanib 300 mg/day than placebo (2.4 vs. 15.7 AEx-IPF per 100 patient years) . HoweverFinally, a recent study using prospective data from 242 patients in the placebo groups of three randomized clinical trials showed that patients who used anti-acid treatments (proton-pump inhibitors or histamine-receptor-2 blockers) at baseline had fewer AEx-IPF than those not taking such treatments at baseline (zero vs. nine events after a mean follow-up of 30 weeks) . The autIPF is a progressive disease and all patients will deteriorate over time. However, IPF has a highly variable disease course. Some patients with IPF suffer AEx-IPF, which are associated with high morbidity and mortality and a worsened prognosis. The methodology used to define AEx-IPF has varied across studies, making it difficult to form an accurate understanding of the incidence and clinical implications of AEx-IPF. More data are needed on the impact of AEx-IPF on patients with IPF. Current treatments for AEx-IPF have only limited data to support their effectiveness and supportive therapy remains the mainstay of care. There is emerging evidence that chronic treatment of IPF may reduce the risk of AEx-IPF and clinical trials investigating this are underway.AEx-IPF: Acute exacerbation(s) of idiopathic pulmonary fibrosis; ALAT: Latin American Thoracic Association; ATS: American Thoracic Society; BAL: Bronchoalveolar lavage; DLco: Diffusing capacity of the lung for carbon monoxide; ERS: European Respiratory Society; FGFR: Fibroblast growth factor receptor; FVC: Forced vital capacity; GERD: Gastroesophageal reflux disease; HRCT: High-resolution computed tomography; IPF: Idiopathic pulmonary fibrosis; JRS: Japanese Respiratory Society; KL-6: Krebs von den Lungen-6; MMP: Matrix metalloproteinase; NHLBI: National Heart Lung and Blood Institute; PaO2: Arterial oxygen tension; PDGFR: Platelet-derived growth factor receptor; PMX: Polymyxin B-immobilized fiber column; TGF: Transforming growth factor; UIP: Usual interstitial pneumonia; VEGFR: Vascular endothelial growth factor receptor.DSK has acted as a consultant, steering committee member, and advisory board member for Boehringer Ingelheim, which is developing nintedanib as a treatment for IPF. The article processing charge for this article has been covered by Boehringer Ingelheim.The author was fully responsible the content of this review article.


Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins.Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from  Pseudomonas aeruginosa, there is an urgent requirement for novel approaches to the treatment of bacterial infections. Lectin-like bacteriocins are highly potent protein antibiotics that display an unusual ability to kill a select group of bacteria within a specific genus. In this work, we show how the lectin-like protein antibiotic, pyocin L1, can kill Pseudomonas aeruginosa with extraordinary potency through specific binding to the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide. The CPA is predominantly a homopolymer of the sugar d-rhamnose that although generally rare in nature is found frequently as a component of the lipopolysaccharide of members of the genus Pseudomonas. The targeting of d-rhamnose containing polysaccharides by pyocin L1 and a related lectin-like protein antibiotic, putidacin L1, explains the unusual genus- specific killing activity of the lectin-like bacteriocins. As we learn more about the link between changes to the microbiome and a range of chronic diseases there is a growing realisation that the ability to target specific bacterial pathogens while maintaining the normal gut flora is a desirable property for next generation antibiotics.Due to rapidly increasing rates of antibiotic resistance observed among Gram-negative pathogens, such as  Clostridium difficile infection The ability to target a subgroup of pathogenic bacteria in a complex bacterial community has potential applications in medicine and agriculture where the maintenance of a ‘normal’ microbiome is beneficial. For example, the use of broad spectrum antibiotics to treat bacterial infections is known to cause a range of complications associated with collateral damage to the microbiome, including antibiotic associated diarrhea and P. aeruginosa and the closely related colicins of E. coliP. aeruginosa, whereas colicins show activity against E. coli and some strains of closely related bacteria such as Salmonella spp. 12 uptake P. aeruginosa there is an urgent requirement for the development of novel therapeutic options since its ability to form drug-resistant biofilms in combination with the presence of an outer membrane that is highly impermeable to many classes of antibiotics can make this pathogen essentially untreatable in some groups of patients. This is exemplified in cystic fibrosis patients where chronic lung infection with P. aeruginosa is the leading cause of mortality In contrast to the broad spectrum antibiotics that are widely used in medicine and agriculture, protein antibiotics known as bacteriocins often target a specific bacterial species or a group of closely related bacterial species P. putida (putidacin L1 or LlpABW) P. syringae (LlpAPss642) and P. fluorescens (LlpA1Pf-5) have been characterised and have the unprecedented ability to kill strains of a broad range of bacterial species within the genus Pseudomonas, but are not active outside this genus Xcm761 from Xanthomonas citri pv. malvacearum LMG 761 has the ability to kill various species within the genus XanthomonasAn interesting addition to this group of protein antibiotics is the recently discovered lectin-like bacteriocins that contain two carbohydrate-binding domains of the monocot mannose-binding lectin (MMBL) family Galanthus nivalis agglutinin (GNA) was the first to be characterised G. nivalis or Allium sativum, it is now recognised that proteins of this class are distributed widely throughout prokaryotes and eukaryotes, where they have evolved to mediate diverse functions Lectins are a structurally and evolutionarily diverse class of proteins produced widely by prokaryotes and eukaryotes and are defined by their ability to recognise and bind carbohydrates. This binding is generally highly specific and mediates a range of diverse functions, including cell-cell interaction, immune recognition and cytotoxicity Pseudomonas spp. show complete conservation of two sugar binding motifs on the C-terminal domain and partial conservation of two sites on the N-terminal domain et ald-mannose or a range of mannose containing oligosaccharides. However, Kds for these protein-carbohydrate complexes were reported in the range from 46 mM for methyl-α-d-mannoside to 2 mM for a mannose containing pentasaccharide The lectin-like bacteriocins are not proteolytically processed and thus consist of a single peptide chain, containing tandem β-prism domains. Sequence alignments of members of this class from P. aeruginosa, and show that it utilises lipopolysaccharide (LPS) as a surface receptor, specifically targeting the common polysaccharide antigen (CPA) that is a conserved homopolymer of d-rhamnose. Structural and biophysical analysis shows that the C-terminal carbohydrate binding motifs are responsible for d-rhamnose recognition and that these sites are specific for this sugar over d-mannose. Further to this, we show that the previously described putidacin L1 also selectively binds LPS from susceptible, but not from resistant, P. syringae isolates and shows selectivity for d-rhamnose over d-mannose. This work shows that the physiologically relevant ligand for the QxDxNxVxY carbohydrate binding site of the lectin-like bacteriocins is indeed d-rhamnose and not d-mannose as previously thought. As such, the genus-specific activity of lectin-like bacteriocins from Pseudomonas spp. can be attributed to the widespread inclusion of the rare d-rhamnose in the LPS of members of the genus Pseudomonas.Despite progress in our understanding of the structure and host range of MMBL-like bacteriocins, the mechanism by which these bacteriocins target susceptible strains and exert their antimicrobial effects is unknown. Here we report on the discovery of a novel member of this family, pyocin L1 from P. aeruginosa infections, we searched the genomes of 10 recently sequenced clinical and environmental isolates of P. aeruginosa for genes with homology to known bacteriocins. One putative bacteriocin gene identified in strain C1433, an isolate from a patient with cystic fibrosis, encodes a protein with 31% identity to the lectin-like bacteriocin LlpA1Pf-5, from P. fluorescens. This protein, designated pyocin L1, contained 256-amino acids with a predicted molecular mass of 28413 Da. Alignment of the pyocin L1 protein sequence with other lectin-like bacteriocins, LlpA1Pf-5, LlpAPss642, putidacin L1 (LlpABW), LlpAAu1504 from Burkholderia cenocepacia and LlpAXcm761 from Xanthomonas citri pv. malvacearum shows that pyocin L1 contains tandem MMBL domains with three conserved QxDxNxVxY MMBL sugar-binding motifs  also known as A-band LPS P. aeruginosa produce two distinct LPS-types that differ in their O-antigen, but share the same core oligosaccharide. The CPA is predominantly a homopolymer of d-rhamnose and the O-specific antigen contains a heteropolymeric repeating unit that varies widely among strains wbpZ, we found that production of CPA, as determined by immunoblotting with a CPA-specific monoclonal antibody P. aeruginosa PAO1, which is sensitive to pyocin L1, with insertions in the genes responsible for the transport of CPA to the periplasm wzt and wzm, encode the ATP-binding component and membrane component of a CPA dedicated ABC transporter wzm and wzt  and analysed the pyocin-CPA interaction by isothermal titration calorimetry (ITC). Titration of pyocin L1 into isolated LPS-derived polysaccharides (a mixture of CPA and the O-specific antigen containing polysaccharides) from PAO1 gave rise to strong saturable exothermic heats of binding  was performed. Titration of pyocin L1 into d-rhamnose gave rise to weakly saturable heats of binding that are significantly larger than the heats observed on titration of pyocin L1 into an identical concentration of d-mannose  spectra on addition of d-rhamnose or d-mannose. In the absence of added monosaccharide 15N-HSQC spectra of pyocin L1, which should contain one crosspeak for each non-proline amide NH as well as peaks for the NH groups in various side chains, were well resolved and dispersed, indicative of a folded protein. Chemical shift perturbation monitored by 15N-HSQC allows the mapping of changes to a protein that occur on ligand binding. Addition of either d-rhamnose or d-mannose up to a concentration of 100 mM did not give rise to large or global changes in chemical shifts  and the structural basis of the d-rhamnose specificity of pyocin L1 we determined the X-ray structures of pyocin L1 with bound d-mannose, d-rhamnose and in the unbound form . The DALI server searches the protein data bank (PDB) to identify proteins structurally related to the query structure In an attempt to determine the location of the pyocin L1 und form . Pyocin und form . This inund form . The oveund form . The higd-mannose and d-rhamnose soaked crystals show clear density for sugar moieties in both sites, C1 and C2  and D180A (C2) variants were performed by titrating protein at a concentration of 100 µM into a solution of LPS-derived polysaccharide (1 mg ml−1) from strain PAO1 (d of 0.15 (±0.07) µM for the wild-type pyocin L1-CPA complex. For both the D150A (C1) and D180A (C2) variants, affinity for CPA was reduced. For the pyocin L1 D150A-CPA complex a Kd of 1.52 (±0.51) µM was determined, a 10-fold increase in Kd relative to the wild-type pyocin L1-CPA complex. However, CPA binding to the D180A variant was severely weakened and although heats of binding were still observed the Kd for this complex, which could not be accurately determined, is likely >500 µM. We also produced a double mutant in which both D150A and D180A mutations were present. For this double mutant, no binding to CPA was observed by ITC. These data show that both the C1 and C2 sugar binding motifs are required for full CPA binding, but that the C2 binding site is the major CPA binding determinant. The killing activity of these sugar binding motif variants showed a good correlation with their ability to bind the CPA. Both the D150A and D180A variants showed reduced cytotoxicity against PAO1 relative to pyocin L1, with the D150A showing a greater reduction in activity and for the D150A/D180A variant very low levels of cytotoxicity were observed . Binding of putidacin L1 to the isolated LPS-derived polysaccharides of the above mentioned strains was tested by ITC. Large saturable heats of binding were observed for putidacin L1 and the LPS-derived polysaccharides from LMG 5456 and LMG 2222, while no binding was observed between putidacin L1 and the LPS-derived polysaccharides from LMG 1247, 2563 or DC3000  of pyocin L1 and putidacin L1 is identical. This observation combined with the specificity of putidacin L1 for d-rhamnose, strongly suggests that it also binds to a d-rhamnose containing O-antigen. Indeed branched d-rhamnose O-antigens are common in P. syringaeAlthough -mannose . Interesobserved . It is id-rhamnose containing O-antigens are utilised as surface receptors for lectin-like bacteriocins from Pseudomonas spp. This is an attractive hypothesis since the inclusion of d-rhamnose in the lipopolysaccharides from members of this genus is widespread and could form an important component of the genus specific activity of this group of bacteriocins.Our data for both pyocin L1 and putidacin L1 indicate that d-rhamnose at the conserved QxDxNxVxY sugar binding motifs of the C-terminal lectin domain. The ability of both pyocin L1 and putidacin L1 to recognise d-rhamnose containing carbohydrates is an important component of their ability to target sensitive strains of Pseudomonas spp. The use of the O-antigen as a primary receptor differentiates the lectin-like bacteriocins from other multidomain bacteriocins such as colicins and S-type pyocins (colicin-like bacteriocins) which utilise outer membrane proteins as their primary cell surface receptors In this work we have shown that pyocin L1 targets susceptible cells through binding to the CPA component of LPS and that primary recognition of CPA occurs through binding of et al. has shown through domain swapping experiments that for putidacin L1 (LlpABW) and the homologous lectin-like bacteriocin LlpA1Pf-5 from Pseudomonas fluorescens, species specificity is governed by the identity of the N-terminal lectin domain d-rhamnose containing O-antigens, with the N-terminal domain interacting with species-specific factors and thus determining the precise species and strain specificity of these bacteriocins. Although there are few clues as to how the lectin-like bacteriocins ultimately kill susceptible cells, we have established a clear role for the C-terminal MMBL domain of these proteins. The roles of the N-terminal MMBL domain and the C-terminal extension remain to be discovered et al, it is clear that all three of these regions are required for killing of susceptible cells.In addition to O-antigen recognition, additional factors, as yet to be determined, are clearly also important in strain and species specificity among the lectin-like bacteriocins. Indeed, recent work from Ghequire P. aeruginosal-form of this sugar that is found in nature. Although otherwise rare, d-rhamnose is found frequently as a component of the LPS of plant pathogens and plant associated bacteria such as P. syringaeP. putidaXanthomonas campestrisBurkholderia spp. E. coli, Salmonella and Klebsiella. It is interesting to speculate that since d-rhamnose is a common component of the LPS of bacterial plant pathogens, that some of the many lectins produced by plants may have evolved to target d-rhamnose as part of plant defence to bacterial pathogens.Interestingly, although rhamnose is frequently a component of plant and bacterial glycoconjugates, such as the rhamnolipids of d-rhamnose containing polymers, indicates that the lectin-like bacteriocins would not interact with either plant or animal cells, since these lack d-rhamnose containing glycoconjugates. In addition, these narrow spectrum antibiotics would leave the majority of the soil microbiome and the gut microbiome of plant-eating animals intact and so would be likely to have minimal environmental impact and minimal impact on animal health. This latter property and the potency of these protein antibiotics could also make the use of lectin-like bacteriocins in the treatment of chronic multidrug-resistant P. aeruginosa infections in humans an attractive proposition.The specificity of lectin-like bacteriocins suggests that these protein antibiotics may be useful in combating plant pathogenic bacteria, either through the use of bacteriocin expressing biocontrol strains or by the production of transgenic plants engineered to express these proteins. The specific targeting mechanism described here, binding of P. aeruginosa were grown in LB at 37°C, P. syringae were grown in King's B Media (KB)  at 28°C.Strains and plasmids utilised in this study are presented in Supplementary P. aeruginosa C1433 pyoL1 gene (ACA GAT CAT ATG AAG TCT CCA AAC AAA AGG AGG) and an XhoI site at the end of the gene (ACA GAT CTC GAG GAC CAC GGC GCG CCG TCG TGG ATA GTC GTG GGG CCA A). The PCR product was ligated into the corresponding sites of the E. coli expression vector pET21a to give pETPyoL1 which encodes pyocin L1 with a C-terminal His6 tag separated from the C-terminus of pyocin L1 by a 6 amino acid linker (RRRAVV). Pyocin L1 was overexpressed from E. coli BL21(DE3)pLysS carrying the plasmid pETPyoL1. Five litres of LB broth was inoculated (1∶100) from an overnight culture and cells were grown at 37°C in a shaking incubator to an OD600 = 0.6. Protein production was induced by the addition of 0.3 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), the cells were grown at 22°C for a further 20 hand harvested by centrifugation. Cells were resuspended in 20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole (pH 7.5) and lysed using an MSE Soniprep 150 (Wolf Laboratories) and the cell debris was separated by centrifugation. The cell-free lysate was applied to a 5-ml His Trap HP column  equilibrated in 20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole (pH 7.5) and pyocin L1 was eluted over a 5–500 mM imidazole gradient. Pyocin L1 containing fractions were identified by SDS PAGE, pooled and dialyzed overnight into 50 mM Tris-HCl, 200 mM NaCl, pH 7.5 and remaining contaminants were removed by gel filtration chromatography on a Superdex S75 26/600 column  equilibrated in the same buffer. The protein was concentrated using a centrifugal concentrator (Vivaspin 20) with a molecular weight cut off of 5 kDa and stored at −80°C until required. The putidacin L1 open reading frame was synthesised (DNA 2.0) and cloned into pET21a via 5′ NdeI and 3′ XhoI restriction sites. The stop codon was removed in order to utilise the pET21a C-terminal His6 tag. Purification of putidacin L1 was performed as for pyocin L1. Constructs to express the pyocin L1 mutants D31A, D97A, D150A and D180A were created using the QuikChange Site Directed Mutagenesis Kit (Stratagene) utilising pETPyoL1 as a template. The primers used were CAA ATT GGT CAT GCA AGC GGC TGG CAA CTT GGT CCT TTA CG and CGT AAA GGA CCA AGT TGC CAG CCG CTT GCA TGA CCA ATT TG for D31A, GCG TAC CTG AAT CTT CAA GAT GCT GGG GAC TTC GGT ATA TTT TC and GAA AAT ATA CCG AAG TCC CCA GCA TCT TGA AGA TTC AGG TAC GC for D97A, CGC CTA GCG TTT CAG GGA GCT GGC AAC CTA GTG ATC TAT C and GAT AGA TCA CTA GGT TGC CAG CTC CCT GAA ACG CTA GGC G for D150A and GAT AGA GCA GTA GTG CAA GAG GCT GGA AAT TTT GTT ATC TAC AAA G and CTT TGT AGA TAA CAA AAT TTC CAG CCT CTT GCA CTA CTG CTC TAT C for D180A. Mutant proteins were purified as described above for wild-type pyocin L1.Pyocin L1 was amplified from the genomic DNA of the producing strain 600 = 0.6 was added to 6 ml of 0.8% soft agar and poured over an LB or KB agar plate. 5 µl of bacteriocin at varying concentrations was spotted onto the plates and incubated for 20 h at 37 or 28°C.Soft agar overlay spot plates were performed using the method of P. aeruginosa E2 (OD600 = 0.6) was centrifuged and resuspended in 100 µl of LB, to which 100 µl (8 mg ml−1) of purified pyocin L1 was added. The culture was grown for 1 h, plated onto a LB agar plate and incubated for 20 h at 37°C. Isolated colonies were identified as P. aeruginosa using 16S PCR as described previously 1.5 ml of a culture of P. aeruginosa E2 and derived pyocin L1 tolerant mutants were sequenced at the Glasgow Polyomics Facility, generating paired-end reads on an Illumina MiSeq Personal Sequencer. Reads were mapped to the previously sequenced parent genomes of P. aeruginosa E2 using the CLC genomics workbench, MAUVE and RAST to create an ordered annotated genome. The CLC genomics workbench was used for genome comparisons and the identification of SNPs/INDELs.The genomes of P. aeruginosa and P. syringae strains as described previously, with modifications including the omission of the final trifluoroacetic acid hydrolysis and chromatography steps P. aeruginosa and P. syringae respectively, pelleted by centrifugation at 6000 g for 20 min, and resuspended in 50 mM Tris, pH 7.5 containing lysozyme (2 mg ml−1) and DNase I (0.5 mg ml−1). Cells were lysed by sonication and the cell lysate was incubated at 20°C for 30 min before EDTA was added to a final concentration of 2 mM. An equal volume of aqueous phenol was added and the solution was heated at 70°C for 20 min, with vigorous mixing. The solution was then cooled on ice for 30 min, centrifuged at 7000 g for 20 min and the aqueous phase extracted. Proteinase K was added to a final concentration of 0.05 mg ml−1 and dialysed for 12 h against 2×5 L H2O. LPS was pelleted by ultracentrifugation at 100,000 g for 1 h, resuspended in H2O and heated to 60°C for 30 min to remove residual proteinase K activity. LPS-derived carbohydrates were isolated by heating LPS in 2% acetic acid for 1.5 h at 96°C. Lipid A was removed by centrifugation at 13,500 g for 3 min followed by extraction with an equal volume of chloroform. The aqueous phase was then lyophilised.LPS was purified from 1 litre cultures of Purified LPS from wild-type and mutant samples were resolved by electrophoresis on 12% SDS-polyacrylamide gels. The LPS banding patterns were visualised by the Invitrogen ultrafast silver staining method. For immunoblotting LPS was transferred onto nitrocellulose membranes and western immunoblotting was performed as previously described using the CPA-specific monoclonal antibody N1F10 and alkaline phosphatase-conjugated goat anti-mouse Fab2 as the secondary antibody d-rhamnose, d-mannose or l-rhamnose were used as titrants and reactions were performed in 0.2 M sodium phosphate buffer, pH 7.5. d-rhamnose (>97%) was obtained from Carbosynth Limited (UK) and d-mannose and l-rhamnose (>99%) from Sigma-Aldrich (UK). For O-antigen-pyocin L1 binding reactions, pyocin L1 or pyocin L1 variants were used as titrant at 100 or 150 µM with cleaved O-antigen sugars dissolved at 1 mg ml−1 in the chamber. For curve fitting we estimated the molar concentration of LPS-derived CPA containing carbohydrate chains at 20 µM based on an estimated average molecular weight of 10 kDa for CPA containing polysaccharides and estimating the percentage of total LPS represented by CPA containing carbohydrates as 20% of the total by weight d. For O-antigen-putidacin L1 binding reactions, O-antigen was used as the titrant at 3 mg ml−1 with 60 µM putidacin L1 in the chamber. Reactions were performed in 20 mM HEPES buffer pH 7.5. All samples were degassed extensively prior to the experiments. Calorimetric data were calculated by integrating the area under each peak and fitted with a single-site binding model with Microcal LLC Origin software. The heats of dilution for each titration were obtained and subtracted from the raw data.ITC experiments were performed on a VP-ITC microcalorimeter . For monosaccharide binding, titrations were carried out at 299 K with regular 15 µl injections of ligands into 60–100 µM pyocin L1 or putidacin L1 at 300 s intervals. 50 mM 15N labelled proteins (0.1–0.2 mM) and unlabelled ligands, d-rhamnose and d-mannose (100 mM), on a Bruker AVANCE 600 MHz spectrometer. Protein samples were prepared with and without the sugars present and volumes were exchanged at fixed ratios, making sure the protein concentration remained unchanged. The spectra were processed with Topspin and analysed with CCPNmr analysis NMR chemical shift perturbation analysis of sugar binding by pyocin L1 and putidacin L1 was carried out at 305 K and 300 K respectively. Fast-HSQC spectra −1 was screened for crystallisation conditions using the Morpheus and PGA crystallisation screens (Molecular Dimensions) 2, 0.03 M MgCl2, 0.1 M Tris/Bicine, pH 8.5) and condition 2  from the Morpheus screen were selected for optimisation by vapour diffusion in 24 well plates . Clusters of needles from these trays grew after 3–7 days and were mechanically separated. The un-soaked crystals were from condition 1, while soaked crystals were from condition 2. Un-soaked crystals were looped and directly cryo-cooled to 110 K in liquid nitrogen; D-mannose and D-rhamnose soaked crystals were soaked for 2–12 min in artificial mother liquor containing 4 M d-mannose or 2 M d-rhamnose, before cryo-cooling to 110 K. X-ray diffraction data were collected at the Diamond Light Source, Oxfordshire, UK at beam lines I04, I04-1 and I24. Automatic data processing was performed with Xia2 within the EDNA package Purified pyocin L1 at a concentration of 15 mg mlGalanthus nivalis agglutinin (PDB ID: 1MSA). Initial phases from Balbes were improved via density modification and an initial model was built using Phase and Build from the Phenix package Fo-2Fc and Fo-Fc maps d-mannose (PDB ID: BMA) corresponded best to the density of bound d-mannose. The density in the d-rhamnose complex best corresponded to α-d-rhamnose, for which no PDB ligand exists; a model for α-d-rhamnose was prepared by removing the oxygen from carbon 6 of α-d-mannose and submitting these PDB coordinates to the Prodrg server, which generated the model and modeling restraints d-rhamnose was designated with the PDB ID: XXR.A dataset from an un-soaked pyocin L1 crystal was submitted to the Balbes pipeline along with the amino acid sequence for pyocin L1 −1. Buffer was read before and after each sample and an average of the buffer scattering was subtracted from the sample scattering. The data obtained for each sample were analysed using PRIMUS 2) was used to calculate the molecular weight at I(0) and radius of gyration, Rg, of PyoL1. Ab initio models of the protein in solution were built using DAMMIF SAXS was carried out on the X33 beamline at the Deutsches Elektronen Synchrotron . Data were collected on samples of Pyocin L1 in the range of 0.5–5 mg mlFigure S1Sequence alignment of pyocin L1 and previously reported MMBL-like bacteriocins. Dark blue shading designates sequence identity, light blue designates chemically conserved residues. The three conserved MMBL sugar-binding motifs  and the partially conserved motif (N2) are boxed in red.(JPG)Click here for additional data file.Figure S2Genetics of CPA biosynthesis in P. aeruginosa. (A) CPA operon, annotated with location of P. aeruginosa E2 tolerant mutant (M4 and M11) deletion and PAO1 transposon insertion mutants. (B) Summary of CPA biosynthetic pathway, showing function performed by genes, shown to induce pyocin L1 tolerance or resistance.(TIF)Click here for additional data file.Figure S31H-15N HSQC spectra of 15N-labelled pyocin L1 in presence (red) and absence (black) of 100 mM (A) d-rhamnose and (B) d-mannose, showing distinctive chemical shifts upon addition of associating sugars. Chemical shift changes specific to a small number of cross-peaks illustrates association of the sugars with a small subset of amino acids, which likely correspond to the residues within the binding sites. Analogous changes are observed for d-rhamnose and d-mannose titrations indicative that the same sites are binding both ligands. Greater shift magnitude is observed for d-rhamnose, indicative of a greater affinity towards this monosaccharide. Boxed regions include cross-peaks used for chemical shift perturbation analysis as shown in (TIF)Click here for additional data file.Figure S4Small angle X-ray scattering of pyocin L1. (A) Ab initio model of pyocin L1 computed with DAMMIF overlaid with the crystal structure. (B) Guinier plot of scattering data indicates that the protein is monomeric in solution (I(0) gives a molecular mass of 29.53 kDa) by extrapolation of scattering intensity to zero scattering angle. Radius of gyration is 2.72 nm, indicative of a folded, globular monomeric particle in solution.(TIF)Click here for additional data file.Figure S5d-rhamnose in C1, C2 and N2 binding sites of pyocin L1.Coordination of  (A) Stereo view of d-rhamnose coordination by binding site C1 (A), C2 (C) and N1 (E), from d-rhamnose soak data. Core binding motif residues (blue) and additional residues contributing to the pocket (white) are shown. Omit map density for d-rhamnose in binding site C1 (B), C2 (D), N1 (F) calculated by refinement of data from d-rhamnose soaked crystal with model built from unsoaked crystal. Density for all sites contoured to 0.15e/Å3.(TIF)Click here for additional data file.Figure S6d-rhamnose, compared with d-mannose.Putidacin L1 shows specificity for  (A) ITC isotherm of d-rhamnose (50 mM) titrated into putidacin L1 (0.1 mM). Weakly saturable heats are indicative of binding with modest affinity (Kd ∼5–10 mM). (B) ITC isotherm of d-mannose (50 mM) titrated into putidacin L1 (0.1 mM). Binding is undetectable under reaction conditions.(TIF)Click here for additional data file.Figure S7l-rhamnose.Putidacin L1 and pyocin L1 do not bind  ITC isotherms of l-rhamnose (50 mM) titrated into putidacin L1 (A) and pyocin L1 (B) both at (0.1 mM). Binding is undetectable under these conditions.(TIF)Click here for additional data file.Table S1Strains and plasmids used in this work.(PDF)Click here for additional data file.Text S1References for supplementary information.(DOCX)Click here for additional data file.


Alu comprise 11% of the human genome Gypsy, occupy nearly half of the maize genome Fossilised mobile genetic elements, including Long Interspersed Element-1 (LINE-1 or L1) retrotransposons, comprise at least two-thirds of the human genome Dicer (DCR) and then assembled with Argonaute (AGO) and other proteins into the RNA-induced silencing complex (RISC) that, in turn, produces RNAi against retrotransposon transcripts Retrotransposition requires transcription of an RNA template for DNA-primed reverse transcription. Several cellular defence mechanisms have evolved to hinder this process, including: 1) promoter methylation and heterochromatinisation, 2) degradation of retrotransposon transcripts via RNA interference (RNAi), and 3) host factor prevention or destabilisation of reverse transcription. To describe in detail just one of a myriad of specific inhibitory pathways, repeat associated small interfering RNAs (rasiRNAs) are present in plant, worm, fly, fish, and mouse gametes and, therefore, represent a highly conserved defence against germ line retrotransposition PLOS Genetics, Ciaudo et al. Dicer markedly decreases L1-Tf promoter methylation and increases L1-Tf transcription, translation, and copy number in cultured mESCs. In particular, DCR−/− mESCs accumulate a remarkable 860 L1-Tf copies (greater than five megabases of genomic DNA) per cell over 20 passages, versus 255 copies per cell in DCRFlx/Flx controls, based on SYBR-Green qPCR targeting the L1-Tf 5′UTR. High-throughput small RNA sequencing then confirmed that DCR−/− mESCs were depleted of approximately 22 nt molecules found in wild-type mESCs, immunoprecipitated with AGO2 and aligned to L1-Tf, and therefore resembling rasiRNAs. Hence, LINE-1 activation in DCR−/− mESCs coincides with rasiRNA depletion and is also possibly influenced by ablation of Dicer-mediated LINE-1 promoter methylation.In this issue of Dicer- and AGO2-independent small RNAs were found to “paint” the L1-Tf 5′UTR. Again, assessing L1-Tf transcription and copy number, Ciaudo et al. found that deletion of XRN2 and DGCR8, respective members of the RNA surveillance and Drosha-DGCR8 Microprocessor pathways, led to increased L1-Tf transcription but not copy number amplification. These observations agree with other recent reports of small RNAs immunoprecipitated with DGCR8 and enriched for LINE-1 sequences −/− mESCs −/− mESCs with human Dicer and found that these cells recapitulated wild-type mESC LINE-1 suppression and differentiated normally, unlike DCR−/− mESCs.Intriguingly, a second class of Dicer and obscure rasiRNA detection (−/− mESCs Dicer or the Microprocessor increases LINE-1 mobilisation in cancer cells, with the latter result at odds with data generated here from mESCs. Future advances in high-throughput sequencing and single cell genomics should enable characterisation of endogenous LINE-1 mobilisation events in stem cells and further delineate the multifaceted roles of Dicer and other factors in LINE-1 inhibition.Evidence for a reciprocal relationship between rasiRNA depletion and LINE-1 activation significantly advances our understanding of RNAi-mediated control of retrotransposition during mammalian embryogenesis. These data are also important because they address a longstanding question of why rasiRNAs cannot be consistently detected in mammalian somatic cells: small RNAs generated by RNA surveillance and the Microprocessor may cleave the same pool of precursor LINE-1 mRNAs processed by etection . As Ciau