Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.Botulinum neurotoxins (BoNTs) are produced by  Food-borne botulinum neurotoxin (BoNT) poisoning results in fatal muscle paralysis. But how can BoNT–a large protein released by the bacteria clostridia–survive the hostile gastrointestinal (GI) tract to gain access to neurons that control muscle contraction? Here, we report the complete structure of a bimodular ∼760 kDa BoNT/A large progenitor toxin complex (L-PTC), which is composed of BoNT and four non-toxic bacterial proteins. The architecture of this bacterial machinery mimics an Apollo lunar module, whereby the “ascent stage” (a ∼290 kDa module) protects BoNT from destruction in the GI tract and the 3-arm “descent stage” (a ∼470 kDa module) mediates absorption of BoNT by binding to host carbohydrate receptors in the small intestine. This new finding has helped us identify the carbohydrate-binding sites and the monosaccharide IPTG as a prototypical oral inhibitor, which extends survival following lethal BoNT/A intoxication of mice. Hence, pre-treatment with small molecule inhibitors based on carbohydrate receptor mimicry can provide temporary protection against BoNT entry into the circulation. Clostridium botulinum are the causative agents of the neuroparalytic syndrome of botulism and pose a serious threat for bioterrorism 50 of ∼35 µg/kg body weight bont gene in one of two different gene clusters, the HA cluster or the orfX cluster The seven botulinum neurotoxin serotypes (BoNT/A–G) produced by Structural information of HAs is available for serotypes C and D, such as the crystal structures of HA33 of serotype C (HA33-C) Various structural models have been proposed for the L-PTC. One recent paper suggested a complex composed of BoNT∶NTNHA∶HA70∶HA17∶HA33 in a 1∶1∶2∶2∶3 ratio for L-PTC/A The functional roles of NAPs are also not well defined. We have recently shown that NTNHA shields BoNT against low-pH denaturation and proteolytic attack in the GI tract by forming the minimally functional PTC (M-PTC), and releases it during entry into the general circulation in vitro by carbohydrate receptor mimics. The monosaccharide IPTG also inhibits oral BoNT/A intoxication in mice, providing the first approach for a possible preventive treatment prior to deliberate BoNT poisoning.Here, we report the structure of a ∼760 kDa L-PTC/A using a combination of X-ray crystallography, single-particle EM and three-dimensional reconstruction (3D-EM). We found that L-PTC/A consists of two structurally and functionally independent sub-complexes, the M-PTC and the HA complex. The HA complex is composed of HA70, HA17, and HA33 in a 3∶3∶6 stoichiometry and adopts an extended three-blade architecture, whereas the M-PTC is situated on top of the HA complex platform. BoNT/A absorption is mainly mediated by nine glycan-binding sites on the HA complex that together form multivalent interactions with host carbohydrate receptors on intestinal epithelial cells. HA complex-mediated toxin absorption can be blocked The high toxicity of BoNT/A prevents imaging of the fully active toxin by cryo-EM. So, we began our analysis with negative-staining EM and determined the 3D molecular envelope of L-PTC/A at ∼31 Å resolution . The M-PD3(residues Pro378–Asn626)–HA17–HA33 (termed the mini-HA complex), and the complete HA70–HA17–HA33 (the HA complex). HA17 formed inclusion bodies and heterogeneous soluble aggregates when expressed and purified alone. This is probably due to the large hydrophobic patches on its surface, which are protected by its binding partners within the HA complex. We then systematically analyzed the solution association of these individual proteins and their complexes using analytical ultracentrifugation (AUC), which was performed at pH 2.3 and 7.6 to mimic the physiological conditions in the GI tract  is degraded and not visible in the crystal structure, which is reminiscent of the post-translational nicking of HA70 into ∼25 and ∼45 kDa fragments that occurs physiologically HA70 consists of three domains (D1–3)   of ∼0.93 Å over 232 Cα atoms. The major HA70–HA17 interactions are composed of 13 pairs of hydrogen bonds and salt bridges. In addition, HA70-Phe547 is buried in a hydrophobic region in HA17 composed of Ile18, Ile92, Ala93, Thr96, and Met140  . The strd Met140 .2 and ∼410 Å2 (per molecule), respectively (HA17 simultaneously binds to two HA33 molecules that form a dumbbell-like shape composed of two β-trefoil domains linked by an α-helix. The two pairs of HA17–HA33 interfaces bury a solvent-accessible area of ∼666 Åectively . The twoectively .2 (per molecule) between them  and bury a solvent-accessible area of ∼961 Åeen them . All theeen them . Due to in vitro-reconstituted HA complex at ∼31 Å resolution (CC∼93.1%)  , and fouC domain).The interface is likely composed of Gly399 and Ile400 in HA70 and Val1128, Gly1129, Glu1210, Pro1212, and Asp1213 in HC (pairwise Cα–Cα distance within 15 Å) , which is situated on top of the HA complex, yielding a ∼760 kDa 14-subunit complex . BoNT/A in 15 Å) . Gly399 Kd∼340 nM) at a stoichiometry of one nLoop to one HA70 trimer  in vitro cleavage by trypsin and pepsin  and are resistant to digestive proteases, as shown by in vitro-reconstituted HA complex markedly reduced the TER. This effect was more marked when the HA complex was applied to the cell monolayer from the basolateral side than from the apical side, which needed ∼17 nM and ∼58 nM to achieve a 90% and 70% decrease in TER after 12 hours, respectively  of ∼1.0 mM, ∼1.0 mM, and ∼1.8 mM, respectively, and that it also bound to isopropyl β-D-1-thiogalactopyranoside (IPTG) Kd of ∼0.8 mM. HA70 bound to α2,3- and α2,6-sialyllactose (SiaLac), both with Kd of ∼0.5 mM, and displayed a lower affinity for N-acetylneuraminic acid (Neu5Ac) (Kd∼7.8 mM). There was no overlap between the carbohydrate selectivity of HA70 and HA33.Many human receptors for microbial pathogens or toxins are glycoconjugates. The L-PTC is known to initiate toxin absorption by binding to intestinal cell surface glycans To determine the physiological relevance of these HA–glycan interactions during toxin absorption, we examined their ability to interfere with the HA complex-mediated disruption of Caco-2 TER. Lac, Gal, and IPTG markedly inhibited the TER reduction induced by the HA complex, and showed higher potencies when applied to the apical than to the basolateral compartment . In contTo fully understand the binding specificity, we determined the crystal structures of HA70 in a complex with α2,3- or α2,6-SiaLac . We founIn contrast to the well-defined conformation of Neu5Ac, the Gal–Glc moiety seems to have a larger structural flexibility and is not essential to HA70–glycan recognition. Specifically, α2,3-SiaLac adopts a linear conformation, which is likely stabilized by a Glc-mediated crystal contact with its symmetry mate. However, α2,6-SiaLac binding to the same site adopts a folded conformation in which there is no crystal contact and Glc has no visible electron density  -HA70, WT-HA17, and HA33-D263A/F278A) did not bind to Gal, whereas the HA70-TPRA complex  failed to bind to Neu5Ac . All animal experiments were conducted under the guidelines of the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training.D3 (residues P378–N626), full-length HA17 (residues M1–I146), and full-length HA33 (residues M1–P293) from BoNT/A1-producing C. botulinum strain 62A were cloned into expression vectors pQE30, pGEX-6p-1, pRSFDuet-1, and pET28a, respectively. In addition, HA17 and HA33 were cloned into the bicistronic pRSFDuet-1 for co-expression.The sequences corresponding to full-length HA70 (residues M1–N626), HA70D3 was cloned into pGEX-6p-1 following the N-terminal GST and a PreScission cleavage site. For HA17 and HA33 in the pRSFDuet-1 vector, HA17 was produced with an N-terminal 6×His tag followed by a PreScission cleavage site, while HA33 had no affinity tag. All HA33 or HA70 mutations were generated by QuikChange site-directed mutagenesis (Stratagene).To facilitate protein purification, a 6×His tag followed by a thrombin cleavage site was introduced to the N-termini of HA70, HA17, and HA33. HA70D3 (pGEX-6p-1), and HA33 (pET28a) were expressed alone; (2) HA70 (pQE30) and HA17 (pRSFDuet-1) were co-transformed into bacteria and co-expressed; (3) HA70D3 (pGEX-6p-1) and HA17 (pRSFDuet-1) were co-transformed into bacteria and co-expressed; and (4) HA17 and HA33 were co-expressed using the bicistronic pRSFDuet-1 vector.Four different protein expression schemes were used to produce the individual HAs or HA complexes. (1) HA70 (pQE30), HA70E. coli strain BL21-RIL (DE3) (Novagen). Bacteria were grown at 37°C in LB medium in the presence of the appropriate selecting antibiotics. Expression was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when OD600 had reached 0.7. The temperature was then decreased to 18°C and expression was continued for ∼16 hours. The cells were harvested by centrifugation and stored at −20°C until use.All recombinant proteins were expressed in the D3 and the HA70D3–HA17 complex were purified using Glutathione Sepharose 4B resins  in phosphate-buffered saline, and eluted from the resins after on-column cleavage using PreScission protease.For purification of His-tagged proteins , proteins were bound to a Ni-NTA  affinity column in a buffer containing 50 mM Tris (pH 8.0) and 400 mM NaCl, and subsequently eluted in the same buffer containing 300 mM imidazole. The eluted fractions of each protein were pooled and dialyzed overnight at 4°C against a buffer composed of 20 mM Tris (pH 8.0) and 50 mM NaCl, then the His-tag was removed with thrombin  or PreScission protease (for the HA17–HA33 complex). GST-tagged HA70D3 and the HA70D3–HA17 complex were purified by MonoQ ion-exchange chromatography in 20 mM Tris (pH 8.0) followed by Superdex 200 chromatography in 20 mM Tris (pH 8.0) and 50 mM NaCl for HA70D3 or 20 mM sodium citrate (pH 5.0) and 100 mM NaCl for the HA70D3–HA17 complex. Each protein or protein complex was concentrated to ∼3–6 mg/ml using Amicon Ultra centrifugal filters (Millipore) and stored at −80°C until used for further characterization or crystallization.The following three schemes were used to further purify the proteins. (1) HA70 and the HA70–HA17 complex was purified by MonoQ ion-exchange chromatography  in a buffer containing 20 mM Tris (pH 8.0) and eluted with a NaCl gradient, followed by Superdex 200 size-exclusion chromatography  in 20 mM Tris (pH 8.0) and 50 mM NaCl. (2) HA33 and the HA17–HA33 complex were purified by MonoS ion-exchange chromatography in a buffer containing 20 mM sodium acetate (pH 5.0) and eluted with a NaCl gradient, followed by Superdex 200 chromatography in 20 mM Tris (pH 8.0) and 50 mM NaCl. (3) HA70The purified HA70 was labeled with Alexa Fluor® 488 carboxylic acid, succinimidyl ester (Life Technologies) according to the manufacturer's instructions. The labeled HA70 was further purified by Superdex 200 chromatography in 20 mM Tris (pH 8.0) and 50 mM NaCl. The calculated dye to protein ratio was ∼2 moles of dye per mole of monomeric HA70.D3–HA17 complexes were produced by co-expression and co-purification as described above. To assemble the mini-HA complex (HA70D3–HA17–HA33), the purified HA33 and the HA70D3–HA17 complex were mixed at a molar ratio of ∼2.5∶1 and incubated at 4°C overnight. The excess HA33 was removed by Superdex 200 chromatography with 20 mM Tris (pH 7.6) and 50 mM NaCl. The fully assembled HA complex was reconstituted by mixing the purified HA70 and the HA17–HA33 complex at a molar ratio of ∼1∶1.3. The mixture was incubated at 4°C overnight and the excess HA17–HA33 complex was removed from the mature HA complex by Superdex 200 chromatography with 20 mM Tris (pH 7.6) and 50 mM NaCl. The fluorescence-labeled HA complex was prepared with Alexa Fluor® 488-labeled HA70 and unlabeled HA17–HA33 complex (HA*) or HA17–HA33DAFA complex (HA33DAFA*) using a similar protocol.The HA17–HA33, the HA70–HA17, and the HA70280 were loaded in 6-channel equilibrium cells and centrifuged at 20°C in an An-50 Ti 8-place rotor at the first speed indicated until equilibrium was achieved and thereafter at the second speed. HA33 was analyzed at rotor speeds of 19,000 and 22,000 rpm. The HA17–HA33 and the HA70D3–HA17–HA33 complexes were analyzed at 12,000 and 14,000 rpm. The HA70–HA17 and the HA70–HA17–HA33 complexes were run at speeds of 6,000 and 8,000 rpm. For each sample, data sets for the two different speeds were analyzed independently using HeteroAnalysis software . Three independent experiments were performed for each sample.Sedimentation equilibrium (SE) experiments were performed in a ProteomeLab XL-I (BeckmanCoulter) analytical ultracentrifuge. Purified HA samples were dialyzed extensively against a buffer containing 50 mM Tris (pH 7.6) and various NaCl concentrations, or 50 mM citric acid (pH 2.3) and various NaCl concentrations. Protein samples at concentrations of 0.4, 0.2, and 0.1 unit of ODD3–HA17–HA33 complex were best fit to a model composed of one HA70D3, one HA17, and two HA33 molecules. HA70–HA17–HA33 forms a tight complex containing three HA70, three HA17, and six HA33. Weak dimerization was observed for the mini-HA complex (Kd of ∼23.1 µM) and the full HA complex (Kd of ∼10.9 µM) at pH 7.6 in the presence of 100 mM NaCl, but was not observed at higher ionic strength. The weak oligomerization Kd suggests that the mini-HA and the full HA complex are monomeric under physiological conditions.The AUC data showed that HA33 is predominantly monomeric in solution at pH 2.3 or pH 7.6. HA17–HA33 forms a tight complex at pH 2.3 or pH 7.6, and the data were best fit to a model composed of one HA17 and two HA33 molecules. The HA70–HA17 complex precipitated at pH 2.3 and was therefore analyzed only at pH 7.6. The best fits for HA70–HA17 clearly suggested a complex composed of three HA70 and three HA17 molecules. The data for the HA70in vitro as described above. Negatively stained EM specimens were prepared following a previously described protocol The L-PTC of BoNT/A was obtained from List Biological Laboratories, Inc.  and Miprolab GmbH . The recombinant HA complex was reconstituted 2. Image quality was monitored on the basis of power spectra quality. Particle boxing, CTF correction, initial model generation, 3D refinement, and resolution assessment were all carried out with the EMAN2 package Particle images were acquired using a 4k×4k TVIPS CCD camera on a Tecnai F20 electron microscope (FEI) equipped with a field emission electron source operated at 200 kV, at a nominal magnification of ∼70,000, resulting in a calibrated pixel size of 4.28 Å/pixel on the object scale after binning. The defocus values were set in the range of 1.5–3.2 µm. The electron dosage was ∼40 electrons/ÅD3–HA17–HA33 complex fitted the 3D-EM map very well (cc = 93.1%). There was a slight deviation at the C-terminal domain of HA33 that is located at the tip of the complex, which may be due to the structural flexibility of HA33. For the L-PTC, the densities for the M-PTC and the HA complex could be clearly identified in the 3D-EM reconstruction map, and the density for the HA complex was manually 3-fold averaged using Chimera and EMAN2. Atomic structures of the M-PTC and the HA complex were then docked separately into their 3D-EM densities, with highest cc values of 87.3% and 87.7%, respectively. The docked M-PTC and the HA complex were then merged to generate the complete pseudo-atomic model for the L-PTC.Visualization and rigid-body docking of atomic models into the 3D-EM density maps were performed using UCSF Chimera 2, and 5% (w/v) PEG [poly(ethylene glycol)] 8K. Micro-seeding was used to improve crystal quality. Carbohydrate complexes were obtained when crystals of the HA17–HA33 complex were soaked with 100 mM Gal, Lac, or LacNAc at 18°C overnight. (3) The HA70D3–HA17 complex was crystallized using 0.1 M sodium acetate (pH 4.8), 12% (w/v) PEG MME 2K, and 0.1 M CsCl. (4) The HA70D3–HA17–HA33 complex was crystallized using 0.1 M Tris (pH 8.2), 0.1 M NaCl, and 6% (w/v) PEG 20K.Initial crystallization screens were performed using a Phoenix crystallization robot (Art Robbins Instruments) and high-throughput crystallization screen kits , followed by extensive manual optimization. The best single crystals were grown at 18°C by the hanging-drop vapor-diffusion method in a 1∶1 (v/v) ratio of protein and reservoir, as follows. (1) HA70 was crystallized with a reservoir solution composed of 0.1 M sodium acetate (pH 4.4) and 1.5 M ammonium chloride. Carbohydrate complexes were obtained when HA70 crystals were soaked in the mother liquor supplemented with 100 mM α2,3-SiaLac, α2,6-SiaLac, or Neu5Ac at 18°C overnight. (2) The HA17–HA33 complex was crystallized using a reservoir of 0.1 M MES (pH 6.2), 0.1 M MgClThe crystals of HA70 and its carbohydrate complexes were cryoprotected in their original mother liquor supplemented with 20% (v/v) ethylene glycerol and flash-frozen in liquid nitrogen. Crystals for all the other samples were cryoprotected in 22% (v/v) glycerol with their mother liquors and flash-frozen in liquid nitrogen. X-ray diffraction data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) or Advanced Photon Source (APS). The data were processed with HKL2000 D3–HA17–HA33 complex by Phaser. The structures of the HA17–HA33 and the HA70D3–HA17 complexes were determined by Phaser using partial structures of the HA70D3–HA17–HA33 complex as search models.The structure of HA70 of BoNT/A was determined by molecular replacement software Phaser http://www.pymol.org).The manual model building and refinements were performed in COOT D3 (200 µM) vs. α2,3-, or α2,6-SiaLac (40 mM); HA70D3 (160 µM) vs. Neu5Ac (80 mM); and HA70 (30 µM) vs. nLoop (400 µM). The data were analyzed using the Origin software package provided by the ITC manufacturer. The thermodynamic values reported are the average of three independent experiments . The HA samples were used as the titrand in the cell and the carbohydrates were used as titrants in the syringe. To control for heat of dilution effects, protein samples were dialyzed extensively against the titration buffer  prior to each titration. Carbohydrates and nLoop peptide were dissolved in the same buffer. The pH of the acidic Neu5Ac solution was carefully adjusted to pH 7.6. The following concentrations were used for pair-wise titrations: HA33 (200 µM) vs. carbohydrates  (50 mM); HA702PO4, 2.5 mM CaCl2, 1.3 mM MgCl2, 20 mM Hepes, and 11 mM D-glucose). The trypsin∶sample ratios (w/w) were 1∶10 (pH 6.0) or 1∶20 (pH 7.5). The digestions were stopped by adding 1 mM PMSF and boiling the samples in reducing SDS-loading buffer for 10 minutes. The pepsin digestions were performed at a 1∶100 ratio (w/w) of pepsin∶sample in a buffer containing 50 mM citrate acid  and 300 mM NaCl. Pepsin digestions were terminated by addition of a 1 M Tris-HCl (pH 8.0) stock solution to give a final concentration of 200 mM and samples were then boiled in the reducing SDS-loading buffer. All samples were subjected to SDS-PAGE.The recombinant HA70–HA17–HA33 complex, HA70, the HA17–HA33 complex, and the M-PTC were subjected to limited proteolysis with trypsin and pepsin overnight at room temperature. The trypsin digestions were performed at two different pHs in buffers containing 50 mM sodium phosphate (pH 6.0 or 7.5) and 300 mM NaCl, or in the Krebs-Ringer's solution  at a density of approximately 105 cells cm−2 for flux studies and determination of transepithelial electrical resistance (TER).Cell culture: Caco-2 cells were obtained from the German Cancer Research Center . Cells were cultured in Dulbecco modified Eagle medium  supplemented with 10% fetal bovine serum, 100 U of penicillin per ml, and 100 mg of streptomycin per ml for up to six months. The cells were subcultured twice a week and seeded on BD Falcon Cell Culture Inserts  in the apical and basolateral reservoir, respectively. TER was determined with an epithelial volt-ohm meter  equipped with an Endohm 12 chamber for filter inserts. Filters with cell monolayers were used at day 11 after seeding which is seven days of post confluency. Only filters with an initial resistance of ≥300 Ω cm70-TPRA complex were pre-incubated with the corresponding carbohydrate over night at 4°C in IMDM and diluted to the final concentration with IMDM prior to administration. The TER upon administration of each carbohydrate in the highest concentrations used was checked in the absence of HA and was virtually identical to that of the control without sugars.Carbohydrate inhibition assays: Lac, Gal, IPTG, Neu5Ac, α2,6- and α2,3-SiaLac were dissolved in IMDM, sterile filtered and stored at −20°C. Neu5Ac stock solution was adjusted to pH 7.4. The wild type HA complex (HA wt), fluorescence-labeled HA complex (HA*), or the HA33-DAFA* was administered to the apical or basolateral reservoirs at final concentrations of 58 nM and 17 nM, respectively. After 24 hour of incubation, 200 µl of samples were taken from the apical and the basolateral reservoir. The marker substance was measured in a BioTek Synergy 4 fluorescence spectrophotometer at 495 nm excitation and 519 nm emission wavelengths.Transport measurement: For paracellular transport studies, filters were incubated in IMDM added to the apical (0.5 ml) and basolateral (1.5 ml) reservoir. As marker substance Alexa Fluor® 488 labeled HA* or HAp-values were determined with the GraphPad Prism 5 program .The mouse protection assay was performed following a previously described protocol D3–HA17–HA33, HA70D3–HA17, HA17–HA33, HA17–HA33–Lac, HA17–HA33–Gal, HA17–HA33–LacNAc, HA70–α2,3-SiaLac, and HA70–α2,6-SiaLac have been deposited with the Protein Data Bank under accession codes 4LO4, 4LO7, 4LO8, 4LO0, 4LO2, 4LO1, 4LO3, 4LO5, 4LO6, respectively. EM 3D reconstructions for the L-PTC and the HA complex have been deposited with the Electron Microscopy Data Bank (EMDB) under accession codes EMD-2417 and EMD-2416, respectively.Atomic coordinates and structure factors for HA70, HA70Text S1File contains Figures S1–S16 and Tables S1–S4, with legends.(PDF)Click here for additional data file.


Caspase-12 (CASP12) modulates the susceptibility to sepsis. In humans, the “C” allele at CASP12 rs497116 has been associated with an increased risk of sepsis. Instead, the derived “T” allele encodes for an inactive caspase-12. Interestingly, Eurasians are practically fixed for the inactive variant, whereas in Sub-Saharan Africa the active variant is still common (∼24%). This marked structure has been explained as a function of the selective advantage that the inactive caspase-12 confers by increasing resistance to infection. As regards to both when positive selection started acting and as to the speed with which fixation was achieved in Eurasia, estimates depend on the method and assumptions used, and can vary substantially. Using experimental evidence, we propose that, least in Eurasia, the increase in the frequency of the T allele might be related to the selective pressure exerted by the increase in zoonotic diseases transmission caused by the interplay between increased human population densities and a closer contact with animals during the Neolithic.We genotyped CASP12 rs497116 in prehistoric individuals from 6 archaeological sites from the North of the Iberian Peninsula that date from Late Upper Paleolithic to Late Neolithic. DNA extraction was done from teeth lacking cavities or breakages using standard anti-contamination procedures, including processing of the samples in a positive pressure, ancient DNA-only chamber, quantitation of DNAs by qPCR, duplication, replication, genotyping of associated animals, or cloning of PCR products. Out of 50, 24 prehistoric individuals could finally be genotyped for rs497116. Only the inactive form of CASP12 was found.We demonstrate that the loss of caspase-12 in Europe predates animal domestication and that consequently CASP12 loss is unlikely to be related to the impact of zoonotic infections transmitted by livestock. CASP12) is a cysteine protease that exerts apoptotic functions, inhibits the inflammatory response and modulates susceptibility to sepsis wCC 0.991, wCT 0.999 and wTT 1, with h = 0.11, then, the onset of the selective sweep for the T allele would have been between 51–55 KYA; c) under neutrality, the ages of the T allele is estimated as 943KYA; d) They do not investigate selection from standing variation. To this, we have to add the persistence of the active CASP12 allele in Sub-Saharan Africa.Caspase-12   show a TCASP12 rs497116 in prehistoric individuals from 6 archaeological sites from the North of the Iberian Peninsula (We decided to test this hypothesis by genotyping eninsula  that dateninsula . Faunists) of 1%, and a heterozygous effect parameter (h) of 0.11, s and h taken from CASP12 rs497116. If we assume that selection on rs497116 is unrelated to animal domestications and that s was ∼1% since the origin of the mutation in Africa , we show that, in Europe, it is possible to go from a frequency of 0.4 to >0.99 in less than 250 generations . Therefore, if Neolithic exerted such a selective pressure, it can explain present day allele frequencies at CASP12 rs497116.We first decided to explore if it is actually possible that Neolithic could have substantially shifted the frequencies of sub-Saharan Africans and Eurasians in a time span ranging from the onset of Neolithic to present time. For Africans, analytical formulae see  show thaCASP12  was found in all samples  Although our simulations use a simple demographic model, they allow us to posit the hypothesis of a Neolithic-driven allele frequency shift scenario with some confidence. Thus, we proceeded with the genotyping stage. Out of 50, 24 prehistoric individuals could be genotyped for rs497116  rs497116 , which iCASP12 allele in Europe was already achieved before animal domestication, and consequently, before the introduction of an important number of new zoonotic infections. That figure could be pushed further back, i.e. earlier than 10,000 YBP, given the results obtained from the pre-Neolithic samples. It could be argued that the SJAPL site dates to a late Neolithic period, and that therefore there has been plenty of time for domestication to shape the allele frequency distribution of CASP12. However, the assignation of SJAPL to late Neolithic is based on the associated lithic industry, as SJAPL is a burial site and not a habitation site. Besides, animal domestication was not yet fully developed at this time, as in habitation sites of this period wild animal remains from hunting activities coexist with faunistic domestic remains in a similar percentage on average For the best-represented sample   the miniCASP12 in Europe predates animal domestication, and that consequently CASP12 loss in Eurasians is unlikely to be related to the impact of zoonotic infections transmitted by livestock. It does not look likely either that the ecological change brought about by Neolithic, i.e., anthropogenic modification of the environment affecting the pathogen or pathogen vector's ecology CASP12 rs497116 alleles. Then, which is the selective agent is responsible for the wide distribution of the CASP12 rs497116 alleles? Malaria immediately comes to mind, but previous investigations have also ruled out malaria as a selective agent responsible for the persistence of the active CASP12 allele in Africa Homo sapiens remains could help us bracketing the time point for the expansion of the inactive allele, and thus, we could be able to infer which environmental scenario was responsible for it.In conclusion, we demonstrate that the spread of the inactive allele of 11w and 12w are the viabilities of genotypes AA and Aa respectively, and hs, where 1-hs = w12/w11, h being the heterozygous effect.To assess if the development of agriculture could have driven a shift from a frequency of 0.4 to a present day frequency of 0.8 as a result of selection in Africa we used the following formula CASP12 (see 8) can reach a mean frequency p′ of 0.8 in 200 generations .Using this formula as a recursive algorithm, it can be shown that an allele A with an initial frequency of 0.4, under a selective regime with s = 1% and h = 0.11, as suggested by the literature for We used SFS_CODE We explored two scenarios. One in which the allele remains neutral along its evolutionary time, and a second one, in which the allele holds a selective coefficient s of 1% (additive) since its origin. The command line, embedded within a Perl script, for the neutral scenario was:system (“./sfs_code 4 1 -n 100 100 0 100 -L 1 1 –mutation $time_mut -W 0 –additive -t 0 -a N -TS 0.0175 1 3 -TE 0.0175 1 -TE 0.607 -B 0 -b 0 -Td 0 P 0 0.1 -Td 0.004 P 0 10 -Td 0.005 P 0 1.68202 -TS 0.211074 0 1 -Td 0.211074 P 1 0.170737565 -TS 0.534772 1 2 -Td 0.534772 P 1 0.724763218 -Td 0.534772 P 2 0.162342748 -Tg 0.534772 P 1 33.98 -Tg 0.534772 P 2 62.27 -Tm 0.211074 P 0 1 6.133603671 -Tm 0.211074 P 1 0 1.047236559 -Tm 0.534772 P 0 1 0.741012229 -Tm 0.534772 P 0 2 0.472619025 -Tm 0.534772 P 1 0 0.091696045 -Tm 0.534772 P 1 2 0.41215962 -Tm 0.534772 P 2 0 0.013100056 -Tm 0.534772 P 2 1 0.092321359 -Tm 0.54805 P 0 1 0 -Tm 0.54805 P 0 2 0 -Tm 0.54805 P 1 0 0 -Tm 0.54805 P 2 0 0 –trackTrajectory T 0.6 P 0 L 0 S 0 R 0.3 0.5 -o $fich_out -e $fich_err –popFreq $fich_popfreq”);http://sfscode.sourceforge.net/SFS_CODE/SFS_CODE_home/SFS_CODE_home.html).Note that the program uses a scenario of human evolution based on the demographic parameters used by We consider 4 simulated populations: present day Africa, Europe at time 7,000 years ago (representing a time point when agriculture started in Europe), present day Asia and present day Europe. Their samples sizes are 100, 100, 0 and 100 respectively. Note also that the variable $time_mut is a random number between 0.001 and 0.003, obtained by a Perl script, indicating the time of origin of the mutation.The command line for the scenario under additive selection (s = 1%) was:system (“./sfs_code 4 1 -n 100 100 0 100 -L 1 1 –mutation $time_mut -W 1 10 1 0 –additive -t 0 -a N -TS 0.0175 1 3 -TE 0.0175 1 -TE 0.607 -B 0 -b 0 -Td 0 P 0 0.1 -Td 0.004 P 0 10 -Td 0.005 P 0 1.68202 -TS 0.211074 0 1 -Td 0.211074 P 1 0.170737565 -TS 0.534772 1 2 -Td 0.534772 P 1 0.724763218 -Td 0.534772 P 2 0.162342748 -Tg 0.534772 P 1 33.98 -Tg 0.534772 P 2 62.27 -Tm 0.211074 P 0 1 6.133603671 -Tm 0.211074 P 1 0 1.047236559 -Tm 0.534772 P 0 1 0.741012229 -Tm 0.534772 P 0 2 0.472619025 -Tm 0.534772 P 1 0 0.091696045 -Tm 0.534772 P 1 2 0.41215962 -Tm 0.534772 P 2 0 0.013100056 -Tm 0.534772 P 2 1 0.092321359 -Tm 0.54805 P 0 1 0 -Tm 0.54805 P 0 2 0 -Tm 0.54805 P 1 0 0 -Tm 0.54805 P 2 0 0 –trackTrajectory T 0.6 P 0 L 0 S 0 R 0.3 0.5 -o $fich_out -e $fich_err –popFreq $fich_popfreq”);DNA extraction was done from teeth lacking cavities or breakages. Anti-contamination procedures 5′-CACCATTAGCACCCAAAGCT-3′ as forward primer and oligo 5′-ACATAGCGGTTGTTGATGGG-3′ as reverse primer. The sequence of the Taqman probe was: VIC-5′-GAAGCAGATTTGGGTAC-3′ (Applied Biosystems).We used our standard procedure to quantify the extracted DNA, which consists on measuring the number of molecules of a segment of 113 bp  of HVR-I of mtDNA by means of qPCR . For this, we used oligo For each sample four replicates were performed, each in 30 µl containing 1× TaqMan Universal PCR Master Mix (Applied Biosystems), 5 µM each primer, 10 µM probe, and 10 µl DNA extract (diluted 1/10 with BSA). The cycling conditions were 1 cycle of 50°C for 2 min, 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. in a StepOne Real-Time PCR System.4, 1.4*105 and 1.4*106 molecules/µl for high concentration samples, and a second standard curve for low concentration samples, with 3 points at 1.12*104, 2.24*103 and 448 molecules/µl. Four replicates were used for each dilution point. Typical  values were respectively  for the high concentration curve, and  for the low concentration samples. Finally, at least three “no-template-controls” were included with each experiment. Quantification of results can be seen in For the standard curves, serial dilutions of plasmid pCR2.1-new (3.9 Kb) including an insert of 450 bp (Eurofins MWG/Operon) containing the HVR-I region of interest, are included in each experiment to generate standard curves. Two different standard curves were performed. One with 3 points of 1.4*10CASP12 genotyping was done by direct sequencing of PCR products. Each extract was sequenced twice. The PCRs were performed in 25 µl of reaction mixture containing 10 mM Tris-HCL pH 8.3; 2 mM of MgCl2, 0.1 µM of each dNTP, 0.4 µM of each primer, 10 units of AmpliTaq Gold (Applied Biosystems) and 10 µl of diluted DNA (1 µl of DNA extract in 10 µl of 1 mg/ml BSA). Cycling parameter were 96°C 1 min for 1 cycle, followed by 45 cycles of 95°C 15 s, 48°C 30 s, 72° 30 s and a final cycle of 72°C 10 min, using Forward primer 5′-CAACTATCTTCATAATCGAAA-3′ and Reverse primer 5′-TTTTATAACCACTGAGTATCC-3′. These primers define amplicons of 91 bp (49 bp without the primer sequences). Both forward and/or reverse sequences were obtained using the above primers and Rhodamine chemistry in an ABI310 Genetic Analyzer (Applied Biosystems).The sequencing of the HVR I  was carried out by sequencing 6 overlapping fragments of approximately 100 bp each, as described in http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Sequences were aligned by eye.Cycling parameter were 95°C for 10 min; followed by 40 cycles of 95°C for 10 sec, annealing temperature for 30 sec, 72°C for 30 sec; and 72°C for 10 min. The annealing temperatures of the primers were as follows: 60°C for the A1/A1R primer pair, 58°C for 2F/2R and 4F/4R, 57°C for 1F/1R and 55°C for 3F/3R and 5F/5R (the primer sequences are listed in CASP12 sequencing resultsa) Duplication of For each of the samples, one extract  was sequenced twice from independent reactions with BigDye 1.1 chemistry using the Reverse primer only. Both sequences were checked to be coincident at rs497116.CASP12 PCR products and sequencing of clonesb) Cloning of CASP12 PCR products (see above) comprising rs497116 were cloned  and from each cloning reaction at least 10 colonies were picked up, sequenced and checked for coincident results at rs497116. In this case most clones showed the same haplotype as the original PCR product as expected, although some clones did show some sequence variability outside rs497116. A mean of 1.8 mutations  per sample cloned (∼80 bp) were found in unique clones, which indicates limited post-mortem damage to the aDNA molecules (rather than contamination with exogenous DNA). . see .CASP12 replication in independent laboratoriesc) CASP12 locus, seven samples  were analyzed in duplicate in two different labs . For this, two different teeth from each sample, one for each lab, were extracted and genotyped independently in each lab. Six samples were coincident (TT) and for one sample from SJAPL no PCR product could be obtained in one of the labs.For the d) mtDNA sequencingBecause SNP rs497116 is monomorphic in European populations, to increase the chances of detecting possible contamination mitochondrial DNA sequences were obtained from 33 samples by direct sequencing as depicted in e) Duplication of mtDNA sequencingIn addition, for 9 samples the same extract was analyzed twice for their mtDNA . MtDNA was chosen because its high variability increases the chances to genetically individualize each sample, and therefore to increase the chances to detect (and track) contamination with exogenous DNA.f) Replication of mtDNA sequencingWe assessed further the reproducibility of the results obtained in the Univ. of the Basque Country by replicating the analysis of the six overlapping fragments of mtDNA HVRI of a second tooth from each of 2 prehistoric individuals in the University of La Laguna (Spain) .g) Cloning and sequencing of mtDNA PCR productsTo further corroborate the results, PCR products corresponding to one of these fragments (∼110 bp) of mtDNA HVRI of 24 different samples  were cloned . From each cloning reaction at least 10 colonies were picked up, sequenced and checked for coincident results. In this case most clones showed the same haplotype as the original PCR product as expected, although some clones did show some sequence variability at sites outside key mutations. A mean of 5.8 mutations  per sample fragment cloned (∼100 bp) were found in unique clones, possibly as a consequence of post-mortem damage to the aDNA molecules (rather than contamination with exogenous DNA) see .h) Genotyping of individuals involved in the studyFinally, the mtDNA types of most of the personnel that was in contact with the samples was also obtained see  in orderi) Genotyping of animal remains found at the sitesCanis familiaris) samples, found at the sites the SJAPL and Marizulo respectively, and one chamois (Rupicapra rupicapra) from the Erralla site could be analyzed. None of them produced PCR products after amplification with human mtDNA primers or human CASP12 primers, but one dog and the chamois produced the expected sequence identifying the species after amplification with mammalian-specific cytochrome b primers In addition, two dog  in the populations studied and other world populations. The cytosine (C) from the ancient allele has been substituted in European populations by a thymidine (T), leading to a non-sense mutation and an inactive truncated molecule.(DOC)Click here for additional data file.Table S2Number of samples analyzed and proportion of which were positive.(DOC)Click here for additional data file.Table S3Mitochondrial haplotypes (HVR-I) and CASP12 rs497116 genotypes of researchers in this study.(DOC)Click here for additional data file.Table S4Mitochondrial DNA and CASP12 sequences of the clones from the samples analyzed in the present study (.xls).(XLS)Click here for additional data file.Table S5Summary of quantification, replication and cloning.(DOC)Click here for additional data file.


Sphingosine-1-phosphate (S1P), a sphingolipid metabolite that is produced insidethe cells, regulates a variety of physiological and pathological responses viaS1P receptors (S1P1–5). Signal transduction between cells consists ofthree steps; the synthesis of signaling molecules, their export to theextracellular space and their recognition by receptors. An S1P concentrationgradient is essential for the migration of various cell types that express S1Preceptors, such as lymphocytes, pre-osteoclasts, cancer cells and endothelialcells. To maintain this concentration gradient, plasma S1P concentration must beat a higher level. However, little is known about the molecular mechanism bywhich S1P is supplied to extracellular environments such as blood plasma. Here,we show that SPNS2 functions as an S1P transporter in vascular endothelial cellsbut not in erythrocytes and platelets. Moreover, the plasma S1P concentration ofSPNS2-deficient mice was reduced to approximately 60% of wild-type, andSPNS2-deficient mice were lymphopenic. Our results demonstrate that SPNS2 is thefirst physiological S1P transporter in mammals and is a key determinant oflymphocyte egress from the thymus. Sphingosine-1-phosphate (S1P), a bioactive sphingolipid that is recognized by five Gprotein-coupled receptors (S1P1–5) and plays a key role in angiogenesis, bonehomeostasis and the immune system 17-S1P, an S1P analog, is rapidly degraded in plasma , which indicates that there is an activedegradation pathway in plasma; therefore, the high S1P level in plasma must bemaintained by a continuous S1P supply from S1P-producing cells SPHK1 and SPHK2 produce S1P by the phosphorylation of sphingosine, and S1P isdephosphorylated to regenerate sphingosine by S1P phosphatases (SPPs) and/orextracellular lipid phosphate phosphatases (LPPs) ko157In addition to platelets, erythrocytes and ECs, several other types of cells with theability to secrete S1P have been identified Human and mouse SPNS2 (hSPNS2 and mSPNS2) have high sequence identities withzSpns2 . When hSPNS2 or mSPNS2 were expressed in Chinese hamster ovary (CHO)cells expressing sphingosine kinase (SPHK) 1, the protein localized to theplasma membrane and exported S1P in a manner similar to zSpns2  [28], . MAanalysis , indicatSPNS2-specific siRNAs, the expression ofSPNS2 mRNA decreased to less than 20% of the control, derived from venousand arterial endothelia, respectively. When HUVECs or HPAECs were treated with control. Toinve control .The amol  ) that SPNS2 expressed in endothelial cellsregulates lymphocyte trafficking in mice.During the reviewing process of this manuscript, it was reported  and Spring Bioscience. Antibodies against CD8 (53-6.7), CD4 (RM4-5), B220(RA3-6B2) and CD62L (MEL-14) were from BioLegend, Inc. Bovine serum albumin(fatty acid-free) (BSA) and sphingosine were from Sigma. S1P andCSpns2 genewith a lacZ-Neomycin cassette  byreplacing part of exon 6 and exon 7 of the mouse cassette . This decassette  and 2. Set al.with some modifications MAECs were isolated according to the methods of Kobayashi HUVECs and HPAECs were purchased from Cell Applications, Inc. These cells werecultured following the manufacturer's instructions.Cell suspensions from the thymus were prepared by mincing organs in RPMI 1640medium and then passing the cells through a nylon mesh. Erythrocytes wereremoved from blood samples by incubation in Lysis buffer (BD Biosciences).Isolated cells were labeled with the antibodies and analyzed using GuavaeasyCyte 8HT.CD4 or CD8 single positive cells were purified from the thymus of mice bynegative and positive selection using specific MACS microbeads conjugated toanti-mouse CD4 or CD8 antibodies (Miltenyi Biotech). Briefly, for the separationof CD4 single positive cells, thymus cell suspensions were labeled with CD8MicroBeads and then passed over an LD column. The negatively selected cells weretreated with CD4 MicroBeads and then passed over an LS column. CD4 singlepositive cells were enriched in the positively selected cell fraction. For theseparation of CD8 single positive cells, the opposite of the selection protocolfor the CD4 single positive cell selection was performed; CD4-positive cellswere depleted and CD8-positive cells were collected. The separated cellpopulations were analyzed by flow cytometry, and the purity was more than80%.Chemotaxis assays were performed as described previously, with slightmodifications g for 15 min at room temperatureand washed twice with a mixture of buffer A (20 mM HEPES-NaOH (pH 7.4), 3.3 mMNaH2PO4, 2.9 mM KCl, 1 mM MgCl2, 138 mMNaCl and 1 mg/ml glucose) containing 1% BSA, followed by immediateresuspension in the same buffer. S1P release from erythrocytes was measured asreported previously, with slight modifications 7 erythrocytes/ml) in buffer Acontaining 1% BSA were preincubated for 5 min at 37°C. Assay buffercontaining 0.2 µM [3H]sphingosine (40 nCi/10µl) in buffer A and 1% BSA was then added to each suspension  and incubated at 37°C. After anindicated incubation period, erythrocytes and the assay buffer were separated bycentrifugation at 12,000×g for 5 sec at 4°C. Lipidswere extracted from the supernatant and erythrocytes and developed by HPTLC(Merck) in butanol/acetic acid/water (3∶1∶1 v/v). Radioactive bandswere quantified with a FLA-3000G Bioimaging Analyzer (Fujifilm).Mice were anesthetized, and blood was collected from their hearts using an acidcitrate-dextrose solution (ACD) as an anticoagulant. Erythrocytes were preparedby centrifugation at 100×g for 15 min atroom temperature. Platelets were prepared by centrifugation of PRP at1,000×g for 15 min and washed with buffer Acontaining 1% BSA, followed by immediate resuspension in the same buffer.S1P release from platelets was measured as reported previously, with slightmodifications 8 platelets/ml) in buffer A containing 1% BSAwere preincubated for 10 min at 37°C. Then, 10 µl of thrombin  was added to the mixture, followed by incubation for10 min. After incubation, the platelets and the medium were separated bycentrifugation at 12,000×g for 5 sec at 4°C. An equalvolume of methanol was added to the supernatant, samples were precipitated bycentrifugation at 12,000×g for 5 min at 4°C, and theresulting supernatant was applied to a Cosmospin filter G and analyzed byUPLC-MS/MS.Mouse blood was collected as described above, and then platelet-rich plasma (PRP)was obtained by centrifugation at 100×5 cells/well.HUVECs and HPAECs were cultured in a 6-well plate for 2 days after the siRNAtransfection, when the number of cells was approximately 2×105cells/well. The medium of the cultured ECs was replaced with releasing medium with 1%BSA, 10 mM sodium glycerophosphate, 5 mM sodium fluoride, 1 mM semicarbazide and20 mM HEPES-KOH (pH 7.4)). After 4 hr of incubation at 37°C, 200 µlaliquots of releasing medium were collected, and the cells were removed bycentrifugation at 12,000×g for 5 min at 4°C. An equalvolume of methanol was added to the supernatant, the samples were precipitatedby centrifugation at 12,000×g for 5 min at 4°C andthe resulting supernatant was applied to a Cosmospin filter G and analyzed byUPLC-MS/MS.MAECs were cultured in a 24-well collagen type I-coated plate until confluent,when the number of cells was approximately 1×10o-phthalaldehyde (OPA) modification followed by HPLC analysisaccording to a modified protocol from Min et al.gfor 15 min at room temperature. C17-S1P (30 pmol) was added to each100 µl aliquot of the sample as an internal standard and subjected tolipid extraction in alkaline chloroform conditions. Organs  were homogenized in PBS, and C17-S1P (30 pmol) was addedto each sample. Total lipid in the organs was extracted using the Bligh-Dyermethod 17-S1P(30 pmol) was added to each 100 µl aliquot of sample and subjected tolipid extraction in alkaline chloroform conditions. Extracted S1P wasdephosphorylated with calf intestinal alkaline phosphatase (30 units) for 90 minat 37°C. The resulting sphingosine was extracted with chloroform, dried andresuspended in ethanol. The OPA modification was performed for 1 hr at roomtemperature. After centrifugation of the samples, a 15 µl aliquot of thesample (135 µl) was analyzed by HPLC (Hitachi) with a Cosmosil 5C 18-AR-IIcolumn .Mouse plasma, organs and cells were assessed form/z 380.31 tom/z 264.2. The cone voltage and collision energy were setat 24 V and 14 eV, respectively.The endogenous S1P secreted from ECs and platelets was measured by UPLC-MS/MS.The UPLC-MS/MS analysis was performed with an Acquity ultra-performance liquidchromatography liquid handling system and a Quattro Premier XE triple quadrupolemass spectrometer controlled by MassLynx (version 4.1) MS and chromatographymanager software (Waters). The separation was performed on an Acquity BEH C18analytical column  using amobile phase consisting of eluent A (water/formic acid (100∶0.1 v/v)) andeluent B (acetonitrile/tetrahydrofuran/formic acid (50∶50∶0.1 v/v)).The gradient was as follows: From t = 0 to 0.5 min A/B50∶50, followed from t = 0.5 to 3 min by a lineargradient from A/B 50∶50 to 0∶100, then fromt = 3 to 7 min A/B 0∶100, t = 7to 7.1 min by a linear gradient from A/B 0∶100 to 50∶50 and finallyfrom t = 7.1 to 12 min A/B 50∶50 at a flow rate of0.300 ml/min. A Quattro premier mass spectrometer was used in the positive ionelectrospray mode with a source temperature of 120°C and a desolvationtemperature of 350°C. Nitrogen was used as the nebulizing, auxiliary anddesolvation gas, while argon was used as the collision gas. S1P was monitoredutilizing a Multiple Reaction Monitor (MRM) at SPNS2or negative control siRNA (Ambion) using Lipofectamine RNAiMAX according to themanufacturer's instructions. Experiments were performed at 48 hr after thetransfection of siRNA.At 24 hr before transfection, cells were plated in 6-well plates at a densitythat would allow them to be 50% confluent at the time of transfection.Cells were transfected with 10 nM siRNA to silence human MAECs were seeded on collagen type I-coated glass and confluent cells were fixedwith cold methanol for 10 min. After incubation with blocking buffer (PBScontaining 1% BSA and 1% FBS) for 30 min at room temperature, theantibody against CD31 (MEC 13.3) conjugated with FITC was incubated with thecells for 3 hr at room temperature. After three washes with washing buffer (PBScontaining 0.1% BSA and 0.1% FBS), the cells were mounted withGEL/MOUNT and photographed with BIOREVO (Keyence).2 and 5mM EGTA, and cryoprotected in 30% sucrose containing 2 mMMgCl2. Subsequently, they were frozen in OCT compound andsectioned at 6–7 mm on a cryostat. The slides were rehydrated with PBS,rinsed with 100 mM phosphate buffer (pH 7.3) containing 2 mM MgCl2,0.02% Nonidet P-40 and 0.01% sodium deoxycholate at 4 °C, andthen stained with 100 mM phosphate buffer (pH 7.3) containing 2 mMMgCl2, 0.02% Nonidet P-40, 0.01% sodiumdeoxycholate, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 0.4 mMTris-HCl (pH 7.3) and 20 µg/mL X-gal at 37 °C in the dark. The slideswere then permeabilized with 0.5% Triton X-100 in PBS, treated with3% H2O2 in methanol for elimination of intrinsicperoxidase activity, and stained with CD31 antibody (Spring Bioscience).Histofine simplestain (Nichirei) and the Liquid DAB+ Substrate chromogenSystem (Dako) were used for the detection of the primary antibody.Tissues from SPNS2-heterozygous mice were fixed overnight in 0.2%paraformaldehyde in 100 mM PIPES (pH 6.9) containing 2 mM MgClTotal RNA was extracted from cells using the PureLink RNA Mini Kit (Invitrogen)according to the manufacturer's instructions. The concentration and purityof the RNA were determined spectrophotometrically by measuring the absorbance at260 nm and 280 nm using a NanoDrop (Thermo). The mRNA was reverse transcribedusing the SuperScript VILO cDNA Synthesis Kit (Invitrogen). We also usedfirst-strand cDNA from C57BL/6J mouse tissues obtained from Genostaff Co., Ltd.for RT-PCR and quantitative real-time PCR.in situ hybridization (ISH) were obtained from GenostaffCo., Ltd. Tissues were dissected, fixed with Tissue Fixative (Genostaff),embedded in paraffin using their proprietary procedures and sectioned at 5µm. For ISH, tissue sections were de-waxed with xylene and rehydratedusing an ethanol series and PBS. The sections were fixed with 4%para-formaldehyde in PBS for 15 min and then washed with PBS. The sections weretreated with 30 µg/ml Proteinase K in PBS for 30 min at 37°C, washedwith PBS, re-fixed with 4% para-formaldehyde in PBS, washed again withPBS, and placed in 0.2 N HCl for 10 min. After being washed with PBS, thesections were acetylated by incubation in 0.1 M tri-ethanolamine-HCl (pH 8.0)with 0.25% acetic anhydride for 10 min. After being washed with PBS, thesections were dehydrated through an ethanol series. Hybridization was performedwith probes at 300 ng/ml in Probe Diluent-1 (Genostaff) for 16 hr at 60°C.After hybridization, the sections were washed in 5× HybriWash (Genostaff),equivalent to 5× SSC, for 20 min at 50°C and then in 50%formamide and 2× HybriWash for 20 min at 50°C, followed by RNasetreatment in 50 µg/ml RNase A in 10 mM Tris-HCl (pH 8.0), 1 M NaCl and 1mM EDTA for 30 min at 37°C. The sections were then washed twice with2× HybriWash for 20 min at 50°C, twice with 0.2× HybriWash for20 min at 50°C, and once with 0.1% Tween 20 in TBS (TBS-T). Aftertreatment with 0.5% blocking reagent (Roche) in TBS-T for 30 min, thesections were incubated with anti-DIG AP conjugate diluted 1∶1000 in TBS-Tfor 2 hr at room temperature. The sections were washed twice with TBS-T and thenincubated in 100 mM NaCl, 50 mM MgCl2, 0.1% Tween 20, and 100mM Tris-HCl (pH 9.5). Staining reactions were performed with NBT/BCIP solutionovernight and then washed with PBS. The sections were counterstained withKernechtrot stain solution and mounted with CC/Mount.Paraffin embedded blocks and sections of mouse thymus, kidney and small intestinefor Quantitative real-time PCR was performed using the first strand cDNA as describedabove with the FastStart Master Mix with ROX (Roche Applied Science) using anABI PRISM 7000 sequence detection system. Primers and probes used forquantitative real-time PCR are listed in t-test. We consideredP-values<0.05 to be significant.To analyze statistical significance, we used an unpaired two-tailedStudent's Figure S1Nucleotide and amino acid sequence of mouse SPNS2. Nucleotidesequences of mouse Spns2 (GenBank accession numberNM_153060) are shown together with the predicted amino acid sequences. Thedeleted region in the SPNS2-deficient mice is indicated in the blue box. Thenucleotide sequence used for the in situ hybridizationprobe is shown with a red box. The positions of the primers used for RT-PCRare indicated with arrows. The positions of the intron are indicated witharrow heads. Amino acid residues conserved between mouse and human SPNS2 areindicated with bold letters.(TIFF)Click here for additional data file.Figure S2Phenotype of SPNS2-deficient mice. (A) SPNS2-deficient mice showan eye-open at birth phenotype. Arrowheads indicate the eyes ofSPNS2-deficient mice that are opened. (B) Body weight of mice at 4 weeksold. Body weight in wild-type ,heterozygous  and SPNS2-deficient  mice wasmeasured at 4 weeks. Error bars represent standard error.*P<0.005 compared to ‘WT’. (C)Survival rate of mice. Survival rate in wild-type , heterozygous  and SPNS2-deficient  mice is indicated as the percent of total natalnumber.(TIFF)Click here for additional data file.Figure S3RNA in situ hybridization inmouse tissue sections. Serial sections of thymus were used forthe detection of Spns2 mRNA with an antisenseSpns2 probe (A) and ECs with a CD31 antibody (B).Serial sections of mouse thymus  were treated with antisense(C and D) or sense (E and F) Spns2 probe. The region usedfor the probe is indicated in Supplemental Spns2 probe(G) or ß-actin probe (H). Serial sections of mousekidney were treated with antisense (I) or sense (J) Spns2probe. Cells in which a positive signal was detected with the antisenseprobe are indicated by arrowheads. Serial sections of mouse aorta weretreated with antisense Spns2 probe (K) orß–actin probe (L). a, aorta, b, bloodvessel, c, cava, Bar, 50 µm.(TIFF)Click here for additional data file.Figure S4Relative amount of ABC transporter mRNA in human ECs aftersiRNA-treatment. HUVECs (A) and HPAECs (B) were transfected withtwo siRNAs targeting SPNS2 mRNA (siRNA-1 or 2) or anegative control siRNA (control). Total RNA was isolated, and mRNA levels ofABCA1, ABCB1, ABCC1,ABCG2 and GAPDH were determined byquantitative real time PCR as described in GAPDH. Graphs show the average values fromfour experiments, with error bars representing standard error.(TIFF)Click here for additional data file.Table S1Results of blood analysis of wild-type and SPNS2-deficient mice.Blood was isolated from 4–5 weeks old wild-type (WT) andSPNS2-defecient (KO) mice and used for analysis of indicated bloodparameters. MCV, MCH and MCHC are Mean Corpuscular Volume, Mean CorpuscularHemoglobin and Mean Corpuscular Hemoglobin Concentration, respectively.(DOCX)Click here for additional data file.Table S2Primers and probes used for quantitative real time PCR. Amountof the transcript for each gene (gene) was determined by the Quantitativereal-time PCR using Forward primer, Reverse primer and indicated number ofTaqMan probe (probe) in Roche Universal Probe Library Set.(DOCX)Click here for additional data file.


Parkin is an E3 ubiquitin ligase, mutations in which cause Autosomal Recessive Parkinson's Disease. Many studies aimed at understanding Parkin function, regulation and dysfunction are performed using N-terminal epitope tags. We report here that the use of small tags such as FLAG, cMyc and HA, influence the physical stability and activity of Parkin in and out of cells, perturbing the autoinhibited native state of Parkin, resulting in an active-for-autoubiquitination species. Depending on the study, the tag is to aid detection and visualisation, or the tag is a large protein fusion to aid in solubility and purification  domain of Parkin and a motif in the C-terminus Taking our results into consideration, efforts should be made to ensure the presence of a tag does not interfere with the native behavioural properties of the protein of interest. Results obtained using tagged proteins should be carefully controlled and interpreted with caution, taking into consideration changes to the protein's natural state upon addition of a fusion tag.600 0.4. After reducing the temperature to 16°C, cells were grown to OD600 0.8 before inducing with 25 µM Isopropyl β-D-1-thiogalactopyranoside (IPTG). Cells were harvested, sonicated and clarified before affinity purification using Ni-NTA resin (QIAGEN) and further purification on a gel filtration column following overnight cleavage with the SUMO protease, Ulp1. Proteins were flash frozen and stored in 50 mM Tris pH 8, 200 mM NaCl, 250 µM TCEP and 10% (v/v) glycerol.cMyc, FLAG and HA were cloned directly onto the N-terminus of full length wild type Parkin in the pET SUMO vector using the Phusion® mutagenesis kit (Finnzymes). Tagged proteins were expressed in the same way as wild type Parkin, essentially as described in Proteolytic susceptibility of each Parkin species was tested by adding 0.025, 0.050, 0.100 and 0.250 (w/w) % of Subtilisin A to 10 µg of Parkin in a total reaction volume of 20 µl. Reactions were incubated at 4°C for 3 hours before stopping with 20 µl of SDS-loading buffer. Gels were stained with SimplyBlue (Invitrogen).Thermal stability assays were performed on an iQ5 iCycler (BioRad) in a 96 well plate format using a temperature gradient from 4°C to 100°C with 0.5°C increments. Each 100 µl reaction contained 5 µg of Parkin buffered with 50 mM Tris pH 7.5, 200 mM NaCl and 250 µM tris-2-carboxyethyl phosphine (TCEP). SYPRO Orange (Invitrogen) fluorescent dye was added to each reaction to a 1/200 dilution. Results are plotted in arbitrary units of the differential of the fluorescence divided by the differential of the temperature versus the temperature. The inflection point of the curve relates to the melting temperature of the protein.6-ubiquitin and 4 mM ATP in a reaction buffer consisting of 50 mM Tris pH 7.5, 2 mM DTT, 5 mM MgCl2 and 5% glycerol were mixed with 0.77 µM of each full length Parkin species before incubating at 37°C for 60 min. The 25 µl final reaction volume was stopped with 12.5 µl SDS-loading buffer. Samples were subjected to western blotting as follows: 1.3 µl loaded for visualisation with anti-Parkin  and 6 µl loaded for visualisation with anti-6xHis .Autoubiquitination assays were carried out as described in 6-Ubiquitin mammalian construct was a kind gift from Dr Axel Behrens (London Research Institute). See 6-Ubiquitin and 2 µg of either empty vector or a Parkin containing vector using Effectene reagents (QIAGEN) according to the manufacturers protocol. During the 48 hr incubation, cells were treated with either 10 µM DMSO or 10 µM MG132  for 12 hr. Clarified soluble lysates were collected and protein levels quantified. 100 µg of each sample was loaded onto a gel for western blotting analysis using anti-Parkin  and anti-Actin  as a loading control. His6-ubiquitinated species were affinity purified from soluble lysate containing a total of 7 mg of protein using Ni-NTA magnetic beads (QIAGEN). Samples were analysed by western blot using anti-6xHis .Full length WT Parkin, cMyc-Parkin, FLAG-Parkin and HA-Parkin were cloned into the Gateway® destination vector pcDNA_DEST40 (Invitrogen) following the Gateway® technology manual. The His


Aspergillus fumigatus conidia can cause severe aspergillosis in immunosuppressed people. A. fumigatus produces a large number of secondary metabolites, some of which are airborne by conidia and whose toxicity to the respiratory tract has not been investigated. We found that spores of A. fumigatus contain five main compounds, tryptoquivaline F, fumiquinazoline C, questin, monomethylsulochrin and trypacidin. Fractionation of culture extracts using RP-HPLC and LC-MS showed that samples containing questin, monomethylsulochrin and trypacidin were toxic to the human A549 lung cell line. These compounds were purified and their structure verified using NMR in order to compare their toxicity against A549 cells. Trypacidin was the most toxic, decreasing cell viability and triggering cell lysis, both effects occurring at an IC50 close to 7 µM. Trypacidin toxicity was also observed in the same concentration range on human bronchial epithelial cells. In the first hour of exposure, trypacidin initiates the intracellular formation of nitric oxide (NO) and hydrogen peroxide (H2O2). This oxidative stress triggers necrotic cell death in the following 24 h. The apoptosis pathway, moreover, was not involved in the cell death process as trypacidin did not induce apoptotic bodies or a decrease in mitochondrial membrane potential. This is the first time that the toxicity of trypacidin to lung cells has been reported.Inhalation of  Aspergillus fumigatus has been considered as the most common airborne fungal pathogen. It is a saprophytic fungus that can grow outdoors on different organic materials including cereals, malted barley, packed hay, compost and tobacco. Growth indoors has been reported using water-damaged building materials A. fumigatus spores is, therefore, likely common throughout life. The size of such spores (2 to 3 µm in diameter) allows them to travel easily through the respiratory tract and into lung alveoli. The spores are commonly found in the environment and can exceed 104 spores/m3 in damp buildings or even 10 to 100 times more in waste or compost handling facilities A. fumigatus can be present in hospital buildings A. fumigatus to cause invasive disease are not currently understood For over twenty years, A. fumigatus genome putatively involved in secondary metabolism A. fumigatusin vivo was characterized by disrupting either the gene encoding the non-ribosomal peptide synthase gliP or the gene encoding the transcriptional factor gliZ, both of which are indispensable to gliotoxin biosynthesis. The decrease in virulence of mutant strains, however, has only been demonstrated in non-neutropenic mice suggesting that gliotoxin plays an important role in the pathogenesis in immunosuppressed but not neutropenic patients Previous studies had identified the presence of 26 gene clusters in the A. fumigatus mycoses have been well documented, few data regarding the potential toxicity of other metabolites present on the conidia are available. Fumigaclavine C and three other minor ergot alkaloids, festuclavine, fumigaclavine A and fumigaclavine B have also been shown to be associated with A. fumigatus conidia A. fumigatus grown on different media bear other metabolites including trypacidin, tryptoquivaline, fumitremorgin A 7A. fumigatus spores/m3A. fumigatus at the beginning of the 1960 s Although the gliotoxin toxicity and its putative involvement in A. fumigatus and to determine the cytotoxic effects they induce in lung cells.The objectives of the present study were, therefore, to evaluate the toxicity of the metabolites borne by the spores of Gliotoxin, verruculogen, fumagillin and helvolic acid were purchased from Sigma-Aldrich , fumitremorgin C, pseurotin A and fumigaclavine A from Alexis Biochemicals , sulochrin from BioAustralis Fine Chemicals . All commercial products were used without further purification (purities higher than 90%). The sources of the other secondary metabolites used as authentic standards were: J.W Dorner, National Peanut Research Laboratory, ARS-USDA, Dawson, USA (fumigaclavine C), C. Avendano, Universidad Complutense, Madrid, Spain (fumiquinazoline F), and P.G. Mantle, Imperial College, London, UK (TR2 toxin). All solvents used in extraction, flash chromatography, liquid chromatography-diode array detector (LC-DAD) and liquid chromatography-mass spectrometry (LC-MS) were analytical grade. Water for high performance liquid chromatography was purified by using a Millipore MilliQ purification system.Aspergillus fumigatus NRRL 35693 strain were produced on Czapek yeast extract agar (CYA) and potato dextrose agar (PDA) medium for 14 days at 37°C in the dark. Ten 90 mm plates were prepared for each culture medium. The conidia were harvested as described previously Large amounts of conidia from A. fumigatus from the Pharmacology-Toxicology Laboratory New Collection (NCPT) were examined for their ability to produce trypacidin (A. fumigatus strain (ΔbrlA) vs the conidiation-restored strain (ΔbrlA::brlA). The generation and morphological characterization of both strains have been described previously A. fumigatus strains was measured after culture on autoclaved grains of CapHorn, a variety of common wheat. Wheat grains were moistened with sterile distilled water for 4 days at 4°C (to reach a water activity aW of 0.98) before 20 min sterilization at 120°C. Thirty grams were placed into a 140 mm diameter petri dish and inoculated with 250 µl of a spore suspension containing 2×105 conidia, obtained from a preliminary culture on PDA medium at 37°C for 7 days. The cultures on the grains were incubated at 25°C for 13 days. The A. fumigatus NRRL35693 (NCPT13) strain was used to identify the toxic metabolites produced by the fungus and to produce and purify trypacidin, monomethylsulochrin and questin.Fifteen other fungal strains of ypacidin . The strm/z range of 50 to 800. MS and MS-MS tuning were performed on authentic fumigaclavine C and the MS-MS collision was set at 40%.Chromatographic analyses were performed on a Thermo-Finnigan surveyor HPLC system with a diode array detector (DAD) and a LCQ XP MAX® ion trap mass spectrometer . The LC separation was accomplished with a 150 mm×2.1 mm Luna 5 µm C18 column . A gradient elution was used at a flow rate of 0.2 ml/min with a mobile phase of 16.6 mM acetic acid (A) and acetonitrile (B) under the following conditions: the program initiated with 80/20% (v/v) eluent A/B for 5 min, then eluent B was increased to 50% (v/v) over 25 min, and to 90% (v/v) over 5 min, held constant at 90% (v/v) for 10 min, then finally returned to the initial 20% (v/v) and kept at this level for the last 10 min. Molecules were ionized with an electrospray ionization source in negative (ESI−) and positive mode (ESI+). For the negative mode, the spray needle was set at a potential of 4.5 kV. Capillary voltage and temperature were −15 V and 375°C respectively. For the positive mode, the source voltage was increased to 5 kV whereas capillary voltage was set to 3 V. The capillary temperature remained unchanged. For both modes, sheath gas and auxiliary gas flow rate of nitrogen were at 70 and 40 (arbitrary units). The tube lens offsets were set for negative and positive mode to −45 V and 5 V respectively. Helium was used in the trap as the damping and collision gas. Culture and fraction extracts were analysed in full scan and MS/MS mode with a After extraction of secondary metabolites with chloroform, the raw extract was fractioned by an automated flash chromatography instrument . The fractionation was performed on a normal phase RediSep 12 g column  at a flow rate of 25 ml/min with a linear gradient. The solvent system was composed of 10% acetic acid in toluene as solvent A and 10% acetic acid in ethyl acetate as solvent B. The % of solvent B increased from 0% to 45% within 10 min with a fraction volume of 5 ml. Thirty fractions were collected from the sample extracted in chloroform. The most active fraction was evaporated, dissolved in methanol and fractionated by HPLC-DAD using a 150 mm×4.6 mm Luna C18 5 µm column  in a HPLC chromatograph . The flow rate was set to 1.5 ml/min. A gradient of water with 0.1% acetic acid and acetonitrile was used as mobile phase increasing from 10% to 40% acetonitrile in 18 min, then increasing to 90% within 10 min, maintained for 10 min, before returning to the initial proportions. The fractionation was performed as follows: F1 (1–10 min), F2 (10–15 min), F3 (15–17 min), F4 (17–19 min), F5 (19–21 min), F6 (21–23 min), F7 (23–25 min), F8 (25–30 min), F9 (30–40 min) and F10 (40–46 min).Trypacidin, monomethylsulochrin and questin were purified from the crude extract by a two step semi-preparative HPLC. In the first step, a 250 mm×7.8 mm Modulo-Cart Strategy 5 µm C18 column  was used under isocratic conditions (32% (v/v) acetonitrile, 68% (v/v) 16.6 mM acetic acid) with a flow rate of 4.5 ml/min. Samples enriched in trypacidin and in monomethylsulochrin and questin were obtained. In the second step, the mobile phase was methanol and 16.6 mM acetic acid with a flow rate of 3.5 ml/min. Trypacidin containing samples were chromatographed isocratically with 45% (v/v) methanol and the monomethylsulochrin and questin containing samples at 50% (v/v) methanol. Purified fractions were evaporated before being dissolved in methanol.25°MeOH = 23 400 M−1 cm−1) and questin (ε25°MeOH = 25 704 M−1 cm−1) and 285 nm for monomethylsulochrin (ε25°MeOH = 14 700 M−1 cm−1).The purity of the compounds was confirmed by LC-MS and NMR as described below. The concentration of the samples was measured by reading the absorbance at 286 nm for trypacidin  spectra were generated using a Bruker Avance DRX-600 spectrometer  operating at 600.13 MHz in CDCl3 solution (70–200 µg sample/600 µl solvent in a 5 mm NMR tube).All measurements were obtained using a Bruker cryoprobe TXI at 300 K with tetramethylsilane as reference standard.1H and 2D-NMR including gradient selection (gs)-correlation spectroscopy (COSY), (gs)-heteronuclear single quantum coherence (HSQC) and (gs)-heteronuclear multiple bonding connectivity (HMBC).To confirm the chemical structures, samples were analyzed using 13C chemical shifts were determined from the f1 projection of HSQC and HMBC diagrams, because the low amount of product (70 µg) precluded the direct measurement of a carbon spectrum.The spectral data of the analysed compounds  are described in 2.The human alveolar carcinoma cell line A549 was obtained from the DSMZ-German Collection of Microorganisms and Cell Cultures . Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with added 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere with 5% CO2.The human bronchial epithelial cells (HBEpC) were purchased from Cell Application Inc. . HBEpC are primary cells derived from normal human bronchi. They were cultured in Bronchial Epithelial Cell Growth Medium provided by Cell Application Inc. at 37°C in a humidified atmosphere with 5% CO4 cells/well for A549 and 2×104 cells/well for HBEpC) and cultured for 24 h. The cells were then exposed to toxins for either 24 h or 48 h. Negative and positive controls were obtained by treating cells with respectively the solvent alone or with 10 µM gliotoxin. Blanks were obtained with the absence of cells in the wells. A 3, 2,5-diphenyltetrazolium bromide (MTT) solution (5.0 mg/ml) was added to the wells (15 µl/well), and the plates incubated at 37°C in 5% CO2 for 4 h. The absorbance (Abs) was measured in a microplate reader ‘Infinite 200’  at 570 nm and 650 nm (reference). The % viability was calculated as follow: (Abstoxin – Absblank)/(Absnegative – Absblank) ×100%.Cell viability was determined using the CellTiter 96® Non- Radioactive Cell Proliferation Assay . The assay is based on the cellular conversion of a tetrazolium salt  2,5-diphenyltetrazolium bromide) into a formazan product by mitochondrial succinate dehydrogenase. The formazan product is detected by absorbance at 570 nm. Cells were plated into 96-well flat plates in 100 µl growth medium (1×1050) was calculated using the software SigmaPlot .The toxin concentration needed to reduce cell viability by 50%  was calculated using the software SigmaPlot .Cell lysis was measured using the CytoTox 96® Non-Radioactive Cytotoxicity Assay . 1×105 cells/well) were seeded in 12-well plates and incubated until reaching approximately 70% confluence. The cells were then exposed to different concentrations of trypacidin  or gliotoxin 5 µM for 24 h. Cells were harvested after trypsinization and centrifugation at 450×g for 6 min, washed with phosphate-buffered saline (PBS) and then fixed in 75% (v/v) ethanol for 4 h at −20°C. The cells were collected by centrifugation, washed twice with PBS and suspended in 0.4 ml of a propidium iodide solution containing RNase  for 30 min at 37°C. Flow cytometry was used to sort cells in G0/G1 phase (2 n DNA), S phase (between 2 n and 4 n DNA), G2/M phase (4 n DNA) and sub-G1 particles (<2 n DNA). The stained cells were analysed using a flow cytometer  and the data were calculated using Flowjo software .The cell cycle was investigated using DNA flow cytometry 6, Molecular Probes, Madison, WI) whose accumulation on the inner surface of the inner mitochondrial membrane is driven by ΔΨm. After 2 h and 24 h exposure to 10 and 50 µM trypacidin, the cells were harvested by centrifugation at 450×g for 6 min and incubated in 1 ml of cell suspension buffer  containing 50 nM DiOC6 for 20 min at 37°C. Carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone 40 µM  was used as positive control. The cells were then collected by centrifugation and suspended in 0.3 ml PBS. DiOC6 fluorescence was recorded using a flow cytometer  and the data were calculated using Flowjo software .The mitochondrial transmembrane potential was assessed using 3,3′-dihexyloxacarbocyanine iodide (DiOC2•−), 10 µM dichlorodihydro-fluorescein diacetate  to detect H2O2 and 5 µM 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate  to detect NO. Pro-oxidants were used as positive controls: 100 µM menadione  for DHE and H2DCF-DA, and 2.5 mM S-nitroso-N-acetylpenicillamine  for DAF-FM. Dead cells were stained by adding 300 ng/ml propidium iodide . Cells were harvested by centrifugation and suspended in 0.3 ml PBS before analysis using a flow cytometer . The data were calculated using Flowjo software .A549 cells were treated with 10 µM and 50 µM trypacidin for 1, 2 and 24 h in the presence of oxidation-sensitive fluorescent probes: 5 µM dihydroethidium  to detect superoxide anion , m/z 199 (90%), m/z 211 (60%), m/z 171 (%) and m/z 147 (10%). This metabolite was identified as tryptoquivaline F or J (m/z 444 in LC-MS positive mode. The collision-induced dissociation MS-MS spectrum at m/z 444 led to a fragment at m/z 199 (100%), m/z 228 (80%), m/z 373 (60%) and m/z 171 (20%). This metabolite was identified as fumiquinazoline C (m/z 345 (2 peaks were observed at m/z 301 and m/z 313. The following metabolite (21.79 min) had the UV spectra of monomethylsulochrin and showed a base peak at m/z 345 in LC-MS negative mode (2 spectra displayed m/z 313 (100%) and m/z 181 (40%). Finally, the last compound (22.34 min) gave a typical anthraquinone UV spectrum and showed a base peak at m/z 283 in LC-MS negative mode. These results strongly suggested that this product was questin  was grown under condiogenesis repressive and inducing conditions. Then, the extract from culture was analysed by HPLC-DAD . As longA. fumigatus culture extracts mentioned in Consequently, extracts of conidia from the other wild strain (NRRL 35693) were also analyzed by LC-MS . All theAspergillus fumigatus was determined by LC-MS analysis  and 7.4±2.4 µM (n = 5) indicating that at the same concentration the loss of the cell viability observed after 24 h exposure was effecting cell lysis.The dose-effect study of trypacidin on cell viability and cell lysis was determined on A549 cells after 24 h exposure in culture. The results show a dose-dependence at concentrations ranging from 0.5 to 50 µM . TrypaciWhen the cells were exposed to 50 µM trypacidin for only 1 or 2 h, the cell viability measured after 24 h of culture was the same as that measured when the cells were exposed to the same concentration of trypacidin for 24 h . These r2•−, H2O2 and NO following short  and long (24 h) exposures to 10 µM and 50 µM of the toxin  as shown by propidium iodide staining  was not significantly different from the IC50 obtained with the A549 cells. m) in the A549 cells  essential for its synthesis brlA strain. Since the genes cluster involved in trypacidin biosynthesis has not been yet characterized it was not possible to compare our metabolome data with the transcriptome data previously published A large number of secondary metabolites are potentially produced by A. fumigatus spore extracts and in bioaerosols from compost A. fumigatus. A previous report A. fumigatus strain by Afiyatullov A. fumigatus. The toxicity of those 5 compounds on airway tissues, however, had not been studied until now.Fischer et al. reported that trypacidin and a tryptoquivaline were both found in Trypacidin was found to be the most toxic metabolite borne by the spores in the current study. The fungal precursor metabolites of trypacidin biosynthesis had low toxicity compared with trypacidin. Sulochrin and monomethylsulochrin did not cause cell lysis or reduce cell viability whereas the two anthraquinone derivatives, emodin and questin, reduced only cell viability at respectively 100 µM and 50 µM. These results are partially in agreement with a previous report showing that sulochrin and questin did not trigger any toxic effects on A549 cells at 150 µM using the same MTT assay The toxicity of trypacidin was assayed with A549 alveolar lung cells and human bronchial epithelial cells which are representative of the upper and lower airway respiratory tract, respectively. Toxicity was dose-dependent with both cell types with a 50% maximum effect around 7 µM. Trypacidin initiated a process of cell death within the first hour of exposure leading to cell necrosis within 24 h as shown by the release of the cytosolic enzyme LDH.Aspergillus genus and deoxynivalenol, a trichothecene mycotoxin produced by several Fusarium species, were previously reported to trigger ROS formation within 1 h of exposure in different types of cells. These included a porcine renal proximal tubular cell line Other mycotoxins such as gliotoxin, aflatoxin B1 produced by the ROS and RNS are known to oxidize cell constituents such as DNA, proteins and lipids, leading to cell damage or death.2O2) and nitric oxide (NO) within the first hour of exposure and that their accumulation after 24 h exposure is correlated with cell lysis as shown by the LDH release. It can be noted that O2•− accumulated in A549 cells only when levels of NO and H2O2 dramatically increased. H2O2 is produced from O2•− through superoxide dismutase (SOD) activity. The O2•− accumulation could be due to the inhibition of SOD activity by an exceeding level of H2O2.It was thus of interest to assess whether trypacidin would induce the formation of ROS and RNS in A549 cells. Our results show clearly that trypacidin induces hydrogen peroxide  of atranone C, brevianamide, cladosporin, mycophenolic acid, neoechinulin and sterigmatocystin induced pulmonary inflammation when applied intratracheally in mice Stachybotrys chartarum (30 spores/g body weight) is cytotoxic for lung cells with LDH being released in the bronchoalveolar lavage fluid Accumulation of spores in cavities of the alveoli (about 4 nl) could lead to pneumocytes being exposed to a toxic level of trypacidin. in vivo.Additional work has to be performed in order to know whether trypacidin could similarly induce lung cell necrosis Figure S1NMR spectrum data of questin, monomethylsulochrin, trypacidin, fumiquinazoline C, tryptoquivaline F.1H and two-dimensional nuclear magnetic resonance (2D-NMR) spectra were generated using a Bruker Avance DRX-600 spectrometer operating at 600.13 MHz in CDCl3 solution as described in Materials and Methods.(DOC)Click here for additional data file.Figure S2Toxicity of fractions issued from flash chromatography. The toxic effect of fractions F12, F14, F16, F18, F19, F20 were measured at four different dilutions in PBS-DMSO 12%: 1/10, 1/50, 1/100, 1/500. Ten µl of each fraction were added to wells containing 100 µl of culture medium. The cells were exposed for 24 h before measuring the cell viability using the MTT assay as described in the Materials and Methods.(PDF)Click here for additional data file.Figure S3Sub-fractionation of F16 fraction using RP-HPLC. F16 fraction was evaporated, dissolved in methanol and fractionated using RP-HPLC. Ten fractions were collected as follows: F1 (1–10 min), F2 (10–15 min), F3 (15–17 min), F4 (17–19 min), F5 (19–21 min), F6 (21–23 min), F7 (23–25 min), F8 (25–30 min), F9 (30–40 min) and F10 (40–46 min). Each subfraction was analysed by HPLC-DAD and LC-MS. A) HPLC chromatogram at 270 nm of fraction F16. B) PDA total scan [200–600 nm] chromatogram of subfractions 5–10.(PDF)Click here for additional data file.Figure S4Dose dependent effect of trypacidin on cell viability and cell lysis. 1×104 A549 cells were cultured for 24 h in 96-well plates and then exposed to trypacidin for 24 h. Cell viability and cell lysis were measured as described in Materials and Methods using the MTT and LDH assays. The figure is representative of 5 independent experiments. The toxin concentration triggering 50% toxic effect (IC50) was calculated using the software SigmaPlot.(PDF)Click here for additional data file.Table S1Aspergillus fumigatus strains studied.(DOC)Click here for additional data file.


Cell cycle was arrested at G1 phase by both compounds in 24 hours exposure group, but global hypomethylation occurred earlier than cell cycle arrest, and the hypomethylation status maintained for more than 20 hours. The mRNA expression of p16 was significantly up-regulated by Cr(IV), especially by potassium dichromate, and the mRNA expression of cyclin-dependent kinases (CDK4 and CDK6) was significantly down-regulated. But protein expression analysis showed very little change of p16 gene. Both qualitative and quantitative results showed that DNA methylation status of p16 remained unchanged. Collectively, our data suggested that global hypomethylation was possibly responsible for Cr(IV) - induced G1 phase arrest,but DNA methylation might not be related to up-regulation of p16 gene by Cr(IV).Hexavalent chromium [Cr(IV)], a well-known industrial waste product and an environmental pollutant, is recognized as a human carcinogen. But its mechanisms of carcinogenicity remain unclear, and recent studies suggest that DNA methylation may play an important role in the carcinogenesis of Cr(IV). The aim of our study was to investigate the effects of Cr(IV) on cell cycle progress, global DNA methylation, and DNA methylation of p16 gene. A human B lymphoblastoid cell line and a human lung cell line A549 were exposed to 5–15 µM potassium dichromate or 1.25–5 µg/cm Chromium (Cr) and its compounds are widely used in many industries such as chromate manufacturing, chrome plating, ferrochrome production and stainless steel welding. Chromium can also be found in the environment in the form of airborne particles from automobile catalytic converters. Hexavalent chromium [Cr (VI)] is one of two major forms of Cr, and it is recognized as a human carcinogen. It is estimated that tens of millions of people are exposed to chromium worldwide in-vitro and human investigations about Cr (VI)-induced DNA methylation have been conducted/carried out so far. It was reported that exposure to potassium chromate was able to induce promoter methylation of gpt transgene in Chinese hamster G12 lung cells Some gpt gene in a transgenic Chinese hamster lung cell line with a bacterial gpt reporter gene were different. Partial methylation in the gpt gene was found after soluble Cr (VI) exposure, but no methylation changes after particulate Cr (VI) exposure Previous studies indicated that Cr (VI) could cause cell cycle arrest in HeLa cells, human lung epithelial A549 cells, human lymphoma U937 cells, p53 mutated cells 2Cr2O7) or particulate (PbCrO4) chromate. Cell cycle progression and cell cycle regulatory gene expressions were analyzed in samples treated with Cr (VI), and the global DNA methylation level and methylation status of p16 gene were also detected. The aim of our study is to investigate the relationship between DNA methylation and cell cycle progression, and to compare the difference of cell cycle arresting effects and its possible mechanisms between soluble and particulate Cr (VI).In the present study, a human B lymphoblastoid cell line and a human lung cell line A549 were exposed to different concentrations of soluble  supplemented with 10% fetal bovine serum . A549, a human lung cell line, was kindly provided by Professor Z. Y. Jia  and cultured in RPMI-1640 supplemented with 10% fetal bovine serum . Cells were maintained at 37°C in a fully humidified atmosphere with 5% CO2, and they were subcultured every 2–3 days.2Cr2O7, Sigma, USA) or lead chromate . The concentrations of potassium dichromate were 0 µM, 5.0 µM, 10 µM, and 15 µM, and the concentrations of lead chromate were 0 µg/cm2, 1.25 µg/cm2, 2.5 µg/cm2, and 5 µg/cm2. The exposure time was 2 hours or 24 hours for potassium dichromate, and 4 hours or 24 hours for lead chromate. Solutions of potassium dichromate were prepared by dissolving it in double distilled water, and then sterilized through a 10 ml syringe with a 0.2 µM filter. Suspension of lead chromate was made according to descriptions by Wise et al. Cells were exposed to potassium dichromate  for 30 minutes at 37°C. The number of cells distributed in G1, S, and G2-M phases of the cell cycle was counted on a flow cytometer  using Cell Quest software.Total RNA was extracted from cells using Trizol® reagent  according to manufacturer’s protocols. The purity and concentrations of extracted RNA were determined by a NanoDrop 2000  spectrophotometer.DNA was extracted from cells using DNA extraction Kit for cells and tissues  following the manufacturer’s protocols. DNA from each sample was treated with sodium bisulfite using the EZ DNA Methylation-Gold™ Kit  according to manufacturer's protocol.MethylFlash™ Methylated DNA Quantification Kit  was used for detecting global methylation status of the DNA isolated from Cr (VI) treated samples. All procedures were carried out according to manufacturer's protocol. To determine the relative methylation status of two different DNA samples, simple calculation for percentage of 5-methylcytosine (5-mC) in total DNA can be carried out using the following formula:5′-TTATTAGAGGGTGGGGCGGATCGC-3′ (forward) and 5′-GACCCCGAACCGCGACCGTAA-3′ (reverse), which amplified a 150-bp product. The primer sequence for the unmethylated reaction were 5′- TTATTAGAGGGTGGGGTGGATTGT-3′ (forward) and 5′-CCACCTAAATCAACCTCCAACCA-3′, giving an amplification product of 234 bp. MSP amplification for the p16 gene was carried out in a final volume of 25 µl, 12.5 µl Zymo Taq™ PreMix, 1.2 µl primers, 2 µl DNA template, 9.3 µl ddH2O. Amplification was performed in a PTC-200 DNA Engine Thermal Cycler machine . The PCR conditions for MSP were as follows: hot start at 95°C for 10 min; then 35 cycles  or 64°C (unmethylated gene) for annealing, 50s at 72°C for elongation followed by 5min at 72°C for extension. The methylated (M) and unmethylated (U) control DNA  were used as a positive control for the methylated and unmethylated p16 gene, respectively. Five microliter of each PCR reaction was directly loaded onto a 3% agarose gel, stained with GelRed™ Nucleic Acid Gel Stain  and visualized under UV illumination using the Alpha Innotech FluorChem® FC2 Imager  to determine if there is a visible band and discriminate the size of the PCR products according to bands of the marker.The methylation status of the promoter of the p16 gene was determined by MSP according to the description of Kondo et al. 5′-AGGGGTTGGTTGGTTATTAG-3′ (forward) and 5′-biotin-CTACCTACTCTCCCCCTCTC-3′ (reverse) for PCR amplifying a part of CPG islands, and 5′-GGTTGGTTATTAGAGGGT-3′ for pyrosequencing. PCR reaction was performed in a volume of 25 µl with 1.5 µl converted gDNA, 12.5 µl Taq™ PreMix , 0.8 µl primers, 10.2 µl ddH2O. The amplification condition was as following: denaturating at 95°C for 10 minutes, followed by 45 cycles at 95°C for 30 seconds, at 58°C (B cells) or 62°C (A549 cells) for 40 seconds, at 72°C for 30 seconds and a final extension at 72°C for 10 minutes. Confirmation of PCR product quality was established on 1.5% agarose gel stained with GelRed. Pyrosequencing was performed using the PyroMark Q24 System  according to manufacturer's instructions.Primers were designed by using the PSQ assay design program . The primer sequences of p16 were 5′-GGCACCAGAGGCAGTAACCA-3′ (forward) and 5′-CCTACGCATGCCTGCTTCTACA-3′, (reverse), CDK6 5′-GTGACCAGCAGCGGACAAATAA-3′ (forward) and 5′-AGCAAGACTTCGGGTGCTCTGTA-3′ (reverse), CDK4 5′-TTCTGCAGTCCACATATGCAACA-3′ (forward) and 5′- GGTCGGCTTCAGAGTTTCCAC -3′ (reverse), and GAPDH 5′- GAAGGTGAAGGTCGGAGTC -3′ (forward) and 5′- GAAGATGGTGATGGGATTTC -3′ (reverse). Relative fold change was determined using the comparative Ct method using GAPDH as endogenous control First-strand cDNA was synthesized by reverse transcription of total RNA with PrimeScript® RT reagent Kit . Quantitative PCR was performed in triplicate per sample for each gene using the SYBR Green PCR system  under the following conditions: 95°C for 1 minute and immediately repetitively cycled 40 times through a denaturing step at 95°C for 5s and an annealing-elongation step at 64°C (p16 and CDK6) or 60°C (GAPDH) for 20s. The primer sequences were as follows: p16 Total cell lysates were resolved by SDS-PAGE and transferred to PVDF membranes , and membranes were blocked and incubated with primary antibodies against p16  and tublin  at 4°C over night. Antibody binding was detected with horseradish peroxidase-conjugated antibodies  and enhanced chemiluminescence . Reactive bands were analyzed using Image-Pro Plus program , and the target protein expression was normalized with respect to tublin expression.P<0.05 for two-tailed tests. Statistical analysis was performed with SPSS 11.0 for windows.One-way ANOVA was used to compare the effects among various treatment groups. Where significant differences were found, Dunnett’s multiple comparison test  or LSD  was applied to make post hoc comparison between the means of controls and treatments. Differences were considered significant at 1-phase cells in samples treated with K2Cr2O7 (p<0.01) in a concentration dependent manner, and the percentages of G1-phase cells in samples treated with PbCrO4 were also significantly (p<0.01) higher than those in controls  lower than those in controls. No significant (P>0.05) changes of the percentages of G2-phase cells were observed in both soluble and particulate chromate treatment groups  higher than those in controls (The effects of Cr (VI) on cell cycle were determined in human B lymphoblastoid cells. In 24 hours exposure group, the percentages of G K2Cr2O7 -B increacontrols -D. Convet groups -B, D. Incontrols -C.P>0.05) change of cell cycle of A549 cells was observed in short-term (2 hours or 4 hours) exposure group  arrested at G1 phase by both chromium compounds, and the percentages of G2/M-phase cells decreased significantly  (The effects of Cr (VI) on cell cycle of A549 cells were similar to those of human B lymphoblastoid cells, but there were also some differences between these two cell lines. No significant  -F, H.2Cr2O7 and PbCrO4 could decrease the percentages of 5-mC in total DNA in a concentration dependent manner. Significant (p<0.01) decrease of global methylation levels were observed at all concentrations of both two chromate compounds, compared with that of controls, except of 5 µM of K2Cr2O7 , and the change of global methylation in both cell lines was similar. The global methylation levels of DNA samples from these two cell lines without treatment were below 0.3% . Both K2 K2Cr2O7 . In huma2Cr2O7 nor PbCrO4 could reduce the methylation level of p16 gene, even at the highest concentrations  change was observed after 2 hours exposure, but mRNA levels of p16 in human B lymphoblastoid cells increased significantly (P<0.01) in a concentration dependent manner when exposure time was extended to 24 hours (P<0.01) increase was only observed at moderate and high exposure groups in A549 cells  difference of mRNA levels of p16 was found between samples treated with PbCrO4 for 4 hours and controls. Significant change of mRNA levels of p16 in human B lymphoblastoid cells was only observed at 2.5 µg/cm2 of PbCrO4 after 24 hours exposure (P<0.01) increase was found at moderate and high exposure groups in A549 cells  on mRNA expression of p16 gene. In the K24 hours -A, and s49 cells -C. In thexposure -B, and s49 cells -D.2Cr2O7 in 24-hour exposure groups , and no significant expression change was observed in 2-hour exposure groups (p<0.01) repressed by moderate and high concentrations of particulate chromate (p<0.01), but no significant change was found in 2-hour exposure groups (2 of particulate chromate in 24-hour exposure groups (p<0.01), and no significant change was observed in 2-hour exposure groups  increase of p16 protein expression levels was only observed in human B lymphoblastoid cells exposed to 15 µM of K2Cr2O7 and in A549 cells exposed to 5.0 µg/cm2 of PbCrO4.The expression change of p16 protein in these two cell lines after being exposed to Cr (VI) for 24 hours was detected by west blotting . Our res2Cr2O7) and particulate (PbCrO4) chromate could induce global DNA hypomethylation in human B lymphoblastoid cells and A549 cells, and it was consistent with reports by Wang et al. that chronic occupational chromate exposure also could induce global DNA hypomethylation in chromate manufacturing workers gpt gene while 24-hour exposure to particulate chromate induced no methylation changes.DNA methylation, one of three main types of epigenetic modifications, plays an important role in the regulation of gene expression. Aberrant DNA methylation is believed to be associated with various diseases and developmental disabilities such as cancer and mental retardation, and global hypomethylation and site-specific hypermethylation are common features of human tumors 2/M phase arrest in human lung epithelial A549 cells exposed to 1 µM –25 µM of potassium dichromate. Hayashi et al. 3 could induce G2 phase arrest in U937 and P53 mutated cells after exposure for 24 hours. Our results were consistent with that reported by Stanley et al. 1 phase after being exposed to 10 µM of potassium dichromate for 24 hours. Therefore, the cellular response of cell cycle arrest to Cr (VI) treatment may be dependent on the types of cells.Panayiotidis et al. 1 phase check point is mainly regulated by CDK 4/6, and cyclins D1, D2, and D3, and p16 is a CDK inhibitor targeting CDK 4/6 1-S phase regulators CDK4 and CDK6 in primary and immortalized granulosa cells, but they didn’t compare the difference of the effects of Cr (VI) on cell cycle arrest between soluble and particulate chromates. The difference of inhibiting effects on CDK 4/6 expression between soluble and particulate chromate might be due to the fact that the up-regulating effect of particulate chromate on CDKs inhibitor p16 was weaker than that of soluble chromate. Thus, Cr (VI) could inhibit both mRNA and protein expressions of CDK4/6 genes, and it may contribute to arresting cell cycle at G1 phase.Cell cycle progression is regulated by cyclin dependent kinases (CDKs), cyclins, and CDK inhibitors. G1/S checkpoint response Furthermore, we measured the effects of Cr (VI) on mRNA expression of CDK inhibitor p16. p16 is known to inhibit the formation of cyclin D-CDK 4/6 complex and thereby contribute to the G1 phase cell cycle arrest induced by Cr (VI) may be associated with global hypomethylation in human B lymphoblastoid cells and A549 cells. Cr (VI) could down-regulate mRNA expression of CDK 4/6 genes and up-regulate mRNA expression of p16 gene, but p16 might be not related to growth arrest for little change in protein expression. In addition, other mechanisms rather than methylation change at the promoter of p16 gene may be involved in up-regulating mRNA expression of p16 gene, and suppression of HDAC expression may be one of them.In conclusion, our study suggested that G