Noninfected mice were used as controls. Capsazepine or vehicle was given intraperitoneally for 6 days. Mice were culled on day 7 after infection and blood and spleen cell phenotype and activation were evaluated. Capsazepine decreased circulating but not spleen F4/80+Ly6G+ cell numbers as well as activation of both F4/80+and F4/80+Ly6G+ cells in infected animals. In addition, capsazepine increased circulating but not spleen GR1+ and natural killer (NK) population, without interfering with natural killer T (NKT) cell numbers and blood NK and NKT activation. However, capsazepine diminished CD69 expression in spleen NKT but not NK cells. Infection increased lipid peroxidation and the release of TNFα and IFNγ, although capsazepine-treated group exhibited lower levels of lipid peroxidation and TNFα. Capsazepine treatment did not affect parasitaemia. Overall, TRPV1 antagonism modulates the innate immune response to malaria.Thousands of people suffer from severe malaria every year. The innate immune response plays a determinant role in host's defence to malaria. Transient receptor potential vanilloid 1 (TRPV1) modulates macrophage-mediated responses in sepsis, but its role in other pathogenic diseases has never been addressed. We investigated the effects of capsazepine, a TRPV1 antagonist, in malaria. C57BL/6 mice received 10 Plasmodium and transmitted from person to person through bites of infected mosquitoes. It affects millions of people annually and is a leading cause of child mortality in underdevelopment countries [ Plasmodium. However, elimination of the parasite does not halt the clinical consequences of disease as surviving patients from severe malaria can develop a range of neurological deficits [ Plasmodium for the first time, being essential to the development of an effective acquired immune response [Malaria is an infectious disease caused by intracellular protozoans of the genusountries . Severe ountries , 3. Avaideficits , 5. In tresponse .2O2) [2−) release [Recently, a protective role for transient receptor vanilloid 1 (TRPV1), a nonselective cation channel found on both neuronal and nonneuronal cells, was suggested in bacteria-induced sepsis –10. Inde2O2)  and regu release –14. Oxid release , 16. Plasmodium berghei ANKA.Herein, the role of TRPV1 in malaria was investigated for the first time. We used the TRPV1 antagonist, capsazepine, to assess whether TRPV1 is able to modulate the innate immune response to malaria in animals infected with ad libitum. All procedures were approved by the Ethics Committee of the University of São Paulo and carried out in accordance with the Brazilian society for animal welfare (SBCAL).Inbred male C57BL/6 mice (8 weeks old) were used. Mice were obtained from the animal's facility of the Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo. Mice were kept in a climatically controlled environment and given food and water5 red blood cells (RBCs) infected with Plasmodium berghei ANKA (clone 1.49L) as described by Elias et al. [Malaria was induced by a single intraperitoneal (i.p.) injection of 10s et al. . Parasits et al. , 19). Thn = 5, uninfected (control) group and n = 8, infected group), intraperitoneally from 24 h following infection, for 6 days  group and n = 8, infected group) was used as controls.In order to assess the role of TRPV1 in malaria, animals received the TRPV1 antagonist, capsazepine . Vehiclμm to create a single-cell suspension. Cells were stained with Trypan blue  and assessed for viability in a haemocytometer. Cells (106) were washed, resuspended in flow cytometry buffer  in phosphate buffered saline-PBS ), and stained with directly conjugated monoclonal antibodies : anti-F4/80 FITC, anti-IAb PE, anti-Ly6G PerCP, anti-GR1 Pe-Cy7, anti-CD3 PE and APC, anti-NK1.1 FITC, and anti-CD69 PECy5. Events were acquired on a BD FACSCanto (BD Biosciences-Immunocytometry Systems) and analyzed using FlowJo software (Tree Star Inc.). In order to analyze monocyte/macrophage and neutrophil populations, cells expressing CD3+, CD4+, CD8+, and CD19+ were gated out and the phenotypic F4/80, GR1, and Ly6G lineage markers were evaluated. Results are expressed as representative two-colour dot-plots as well as number of cells (×106), except for IAb, expressed as mean fluorescence.Blood and spleen samples from infected and uninfected mice and single-cell suspensions were prepared. Peripheral blood cells were isolated by Percoll gradient . Spleens were homogenized and passed through a nylon mesh of 70 α, IFNγ, IL-4, IL-6, IL-2, IL-10, and IL-17 were evaluated by using a cytometric bead array (CBA) mouse Th1/Th2/Th17 cytokine kit  according to manufacturer's instructions. Analysis was performed on a Facscalibur cytometer flow cytometer (BD Biosciences-Immunocytometry Systems). Results were calculated in CBA FCAP Array software  as pg/mL and are expressed as fold-increase relative to uninfected controls.The plasma levels of TNFμL of sample were incubated with 100 μL of PBS and 400 μL of thiobarbituric acid , at 90°C for 45 min. Samples were then centrifuged at 1,000 xg for 10 min. Three hundred μL of the supernatant was incubated with 300 μL of butanol  and 30 μL of a saturated solution of sodium chloride . Samples were mixed in a vortex, centrifuged at 1,000 xg for 2 min and then added to a 96-well plate (200 μL/well). Absorbance was read at 535 and 572 nm and the difference between the absorbance was used to calculate the aldehyde concentrations, using the molar extinction coefficient of the chromophore (1.56 × 105 M−1/cm−1). Results are expressed as fold-increase relative to uninfected controls.Total plasma aldehyde  concentrations were quantified as an index of lipid peroxidation and oxidative stress, as previously described , 21. Brit-test when appropriate. The P values < 0.05 were considered significant.The results are presented as the mean ± standard deviation (SD). The percentages of inhibition are reported as mean ± SD of inhibitions obtained in each individual experiment compared with control samples. Statistical comparisons of the data were performed by ANOVA followed by Bonferroni and unpaired +, F4/80+Ly6G+ and Ly6G+ cells. Malaria induction had no effect on Ly6G+ cell population as no statistical significance was found between any of the evaluated groups. Mean ± SD values for Ly6G+ populations are as follows: vehicle-uninfected group 2.0 ± 0.6, capsazepine uninfected group 1.3 ± 0.4, vehicle-infected group 0.9 ± 1.1, and capsazepine infected group 1.3 ± 0.8. On the other hand, P. berghei ANKA infection increased F4/80+Ly6G+ cell numbers in both vehicle- (3.9-fold increase) and capsazepine- (1.9-fold increase) treated groups when compared to uninfected controls, whilst no effects were observed on F4/80+ cell population  and F4/80+Ly6G+ (6.6-fold increase) circulating cells when compared to uninfected animals, as denoted by expression of IAb on these cells . However, infected mice treated with capsazepine exhibited a higher number of these cells when compared with both their uninfected- (1.8-fold increase) and infected- (2.4-fold increase) control animals. Mean ± SD values for GR1+ populations are as follows: vehicle-uninfected group 2.9 ± 0.6, capsazepine uninfected group 2.5 ± 0.4, vehicle-infected group 1.9 ± 0.4, and capsazepine infected group 4.6 ± 0.5  (P < 0.05). Capsazepine treatment in uninfected animals had no effects in regard to expansion or activation of both F4/80+ and F4/80+Ly6G+ cells  and capsazepine-  treated groups when compared to their respective uninfected controls  and capsazepine-  treated groups when compared to their respective uninfected controls  and NKT (CD3+NK1.1+) cells. Peripheral blood NK and NKT cells were detected in all groups of uninfected and infected animals . Production of IL-4, IL-6, IL-2, IL-10, and IL-17 was not detected in any of the evaluated groups.ldehydes . However Figures . TNFα pr malaria . Vehicleα, IL-10, and IL-6 [Since its discovery, evidence has accumulated that TRPV1 has the potential to play a key role in a variety of pathologies, especially those associated with imbalances of the immune and inflammatory response, such as asthma –24 and rand IL-6 –9. So fa in vivo produces a similar profile to that of TRPV1 KO mice in response to bacterial infection [ P. berghei ANKA in a susceptible strain of mice known to develop cerebral malaria-like symptoms and to present with 60–100% mortality within the second week following injection . Our stview see , 22. Plasmodium removal [Evidence suggests that the innate immune response plays an important role during the early phase of infection, with activated monocytes and neutrophils releasing nonspecific inflammatory mediators such as ROS and cytokines , 39 and  removal . As a re removal , 42. Dur removal .+). Interestingly, we detected a group of circulating F4/80+Ly6G+ cells which became markedly expanded and activated (as denoted by IAb expression) following infection. This is the first report to our knowledge of their contribution to malaria. However, it was recently suggested in a model of infection caused by vaccinia virus inoculation in C57BL/6 mice that F4/80+Ly6G+ cells are indeed monocytes with a great capacity of producing ROS and IFNγ whilst Ly6G− monocytes produce NO and TNFα [+Ly6G+ cells replace Ly6G− monocytes as infection progresses. Herein, we show the existence of F4/80+Ly6G+ cells also in the spleen and that they exhibit a similar profile (in terms of number and activation pattern) to that of Ly6G− cells in this tissue. As the population of F4/80+Ly6G+ cells rises, GR1+ cells (myeloid-derived suppressor cells) decline with infection. This is expected as an increase of mature monocytes and neutrophils normally occurs as a result of infection [+, F4/80+Ly6G+, and GR1+ cells could not be noticed at spleen level. However, it is possible that these alterations may occur in this organ at a different time point not addressed by this study.As previously mentioned, TRPV1 can be found on immune cells. It is then expected that antagonism of TRPV1 channels expressed on these cells would affect their phenotypes. Our results show that at 7 days after infection, there is activation of both monocytes and spleen macrophages .α and IFNγ. Indeed, high systemic levels of inflammatory mediators such as TNFα, IFNγ, and aldehydes are correlated with severe malaria in humans [α were detected in animals with malaria that had been treated with capsazepine, IFNγ production remained similar to that observed for samples obtained from vehicle-infected mice. A reduction of oxidative stress was expected as capsazepine was previously suggested to inhibit oxidative stress in cultured RAW264 monocytes/macrophages in a TRPV1 independent manner, although this data was obtained from cells that had not been challenged with any pathogen product [α upon bacterial infection [α. Also, TRPV1 effects on TNFα production may vary at different stages of malaria.To evaluate the impact this shift on monocyte profile had on inflammatory mediator release, we measured the levels of plasma aldehydes (index of lipid peroxidation secondary to oxidative stress) and cytokines such as TNFn humans . In factn humans , 52. In n humans , 54. We  product . Later,  product  and to m product , 56. Hownfection , 9, 32.  Plasmodium-infected RBCs and malarial antigens, in addition to producing IFNγ in order to contain parasitaemia [ P. berghei ANKA-induced infection caused expansion of spleen NKT but not NK cell population. This was not accompanied by any change on circulating NK and NKT cell numbers. It is possible that at this time-point, NK and NKT cells have already migrated to the brain. Indeed, Hansen and collaborators showed that NK cells accumulate into the brain of C57BL/6 mice as early as 4 days following infection with P. berghei ANKA, triggering the migration of T cells to the brain microvasculature [+Ly6G+ monocytes by capsazepine would impair IFNγ release. However, we show capsazepine inhibitory effects on these populations do not affect IFNγ production in malaria, indicating that either TRPV1 may not play a role on IFNγ production or NK cells become the sole source of IFNγ once TRPV1 is blocked. In addition, the lack of effect of capsazepine on parasitaemia may be related to the similar levels of IFNγ detected in both groups of animals.NK and NKT cells are important effectors of the innate immune response to malaria, directly recognizingsitaemia , 18, 40.sitaemia ). In addsitaemia , 57. AlsOur study provides the first evidence that TRPV1 modulates malaria by mediating innate immune response, specifically by interfering with the expansion and activation of effector cells, especially monocytes. We also show that TRPV1 regulates the immunological balance between different monocyte populations in addition to modulating mediator release by them. It is possible that blocking TRPV1 may be either beneficial, as a reduction of oxidative stress may reflect on reduced vascular dysfunction, or deleterious, as impairment of innate response may lead to an inefficient removal of the parasite in addition to an inefficient acquired immune response to malaria. However, the impact TRPV1 antagonism may have on severe malaria outcome is of importance and remains to be investigated.


Down syndrome (DS), caused by trisomy of chromosome 21, is one of the most common genetic disorders. Patients with DS display growth retardation and inevitably develop characteristic Alzheimer’s disease (AD) neuropathology, including neurofibrillary tangles and neuritic plaques. The expression of amyloid precursor protein (APP) is increased in both DS and AD patients. To reveal the function of APP and elucidate the pathogenic role of increased APP expression in DS and AD, we performed gene expression profiling using microarray method in human cells overexpressing APP. A set of genes are significantly altered, which are involved in cell cycle, cell proliferation and p53 signaling. We found that overexpression of APP inhibits cell proliferation. Furthermore, we confirmed that the downregulation of two validated genes, PSMA5 and PSMB7, inhibits cell proliferation, suggesting that the downregulation of PSMA5 and PSMB7 is involved in APP-induced cell proliferation impairment. Taken together, this study suggests that APP regulates global gene expression and increased APP expression inhibits cell proliferation. Our study provides a novel insight that APP overexpression may contribute to the growth impairment in DS patients and promote AD pathogenesis by inhibiting cell proliferation including neural stem cell proliferation and neurogenesis. Down syndrome (DS), caused by trisomy of chromosome 21, is one of the most common genetic disorders. Patients with DS display growth retardation, cardiac and gastrointestinal abnormalities, immune system defects and inevitably develop characteristic Alzheimer’s disease (AD) neuropathology including neuritic plaques and neurofibrillary tangles. Neuritic plaques, the unique feature of AD neuropathology, mainly consist of amyloid-beta protein (Aβ)13456789APP gene, located on chromosome 21, spans approximately 290,586 bp of genomic DNA. It is ubiquitously expressed in a wide variety of human tissues and highly expressed in human brains. APP, a type I transmembrane protein, is predominantly cleaved by α-secretase, generating an N-terminal secreted soluble APPα (sAPPα) and a C-terminal 83-amino acid fragment (C83). C83 is further cleaved by γ-secretase to release a 3 kDa fragment (P3) and APP intracellular domain (AICD)111213in vivo, to generate a C-terminal fragment of 99 amino acids or 89 amino acids (C99 or C89), which will be further cleaved to generate Aβ or truncated Aβ and AICD by γ-secretase151617182021The In addition to Aβ generation and neuritic plaque formation, additional functions of APP have been implicated. For example, APP knockout mice display various abnormalities, such as reduced body weight and size, increased frequency and severity of kainite-induced seizures, and deficits in learning and memory2324252728293032333536384041424344454647APP expression is increased in both DS patients and AD patientsTo investigate the role of APP overexpression in the regulation of global gene expression, the wildtype human APP was stably transfected into HEK 293 cells as HAW cells. Compared with parental HEK293 cells, exogenous full-length APP and C-terminal fragment of APP with 83 amino acids (C83) were detected by C20 antibody which recognizes the last 20 amino acids of APP C-terminal . Whole gIt was reported that the differentially expressed genes in DS patients distribute on all chromosomes, ranging from 3–6.6%, except for 27% genes on chromosome 21To reveal the potential function of differentially expressed genes by APP overexpression in HAW cells, the enrichment analysis of gene ontology (GO) was performed by DAVID, a web-based software for gene expression analysis. 2169 genes were identified by DAVID, including 910 upregulated and 1259 downregualted genes. 197 GO categories of biological processes were significantly affected (p < 0.05), including DNA replication (33 genes), transcription (268 genes), translation (99 genes), cell cycle (117 genes), negative regulation of cell proliferation (57 genes), cell morphogenesis involved in differentiation (40 genes), and regulation of neuron differentiation (24 genes) etc. . SubcateGO enrichment analysis only presents each function independently. Pathway analysis considers the function dependency and molecular interactionTo confirm the altered gene expression by APP in microarray experiments, quantitative PCR following reverse transcription (qRT-PCR) was performed by using three sets of independent samples. As GO enrichment analysis and pathway analysis clearly showed that APP overexpression significantly affected DNA replication, transcription, translation, cell adhesion, cell cycle and cell proliferation, 12 genes involved in these GO categories and pathways were selected to be validated. Data from microarray experiments showed that the expression of SSBP1 (DNA replication), POLR1C (transcription), POLR2C (transcription), RPL4 (translation), EIF2A (translation), PSMA5 (cell cycle), PSMB7 (cell cycle), PSMB10 (cell cycle), PRMT5 (cell proliferation) and CCND1 (cell cycle) was reduced to 0.58, 0.47, 0.55, 0.23, 0.50, 0.52, 0.41, 0.26, 0.25 and 0.42 fold, respectively, while the expression of TP53INP1 (cell cycle arrest and apoptosis) and PCDH19 (cell adhesion) was increased to 6.48 and 53.06 fold. Consistently, qRT-PCR results showed that the expression of SSBP1, POLR1C, POLR2C, RPL4, EIF2A, PSMA5, PSMB7, PSMB10, PRMT5 and CCND1 was reduced to 0.35 ± 0.04, 0.42 ± 0.08, 0.58 ± 0.09, 0.50 ± 0.09, 0.33 ± 0.04, 0.30 ± 0.06, 0.44 ± 0.01, 0.26 ± 0.04, 0.25 ± 0.04 and 0.40 ± 0.08 fold, respectively, while the expression of TP53INP1 and PCDH19 was increased to 5.11 ± 0.69 and 35.35 ± 7.24 fold (P < 0.05) . To furtTo assess the effect of APP on cell growth, the number of cell population doublings (PDs) was examined in both HEK293 and HAW cells, and the growth curve was plotted after 12 day’s culture. The growth rate of the HAW cells was 77.18% of HEK293 cells, 8.80 ± 0.46 PDs vs. 11.40 ± 0.36 PDs (p < 0.05) . To furtTo determine whether APP-induced inhibition of cell proliferation is mediated by the dysregulated genes identified by microarray and qRT-PCR, knockdown experiments were performed in HEK293 cells by using siRNAs of two identified genes involved in cell cycle and cell proliferation, PSMA5 and PSMB7, respectively. Three days after PSMA5 or PSMB7 siRNA transfection, the expression of PSMA5 or PSMB7 was markedly reduced compared with that in scrambled siRNA transfected cells . The BrdDysregulation of gene expression has been implicated in DS1052535455495058In this study we performed whole genome gene expression assay in a human cell line. 3003 genes of 37847 illumina genes are significantly altered, around 8% of total genes, indicating that APP has global effects on gene regulation, not limiting to a small number of genes. Moreover, the affected genes distribute on23 chromosomes, ranging from 4.00% to 9.93%. It suggests that increased APP expression relatively equally affects the gene regulation on all chromosomes with no specific chromosomal preference. In addition, APP overexpression does regulate the expression of genes on chromosome 21 (9 upregulated and 11 downregulated genes). APP overexpression can further increase the expression of 9 genes on chromosome 21, indicating that the expression level of genes on chromosome 21 could increase to more than 1.5 fold in DS. Meanwhile, APP also reduces the expression of another 11 genes on chromosome 21, which may partially explain that the expression level of a group of genes on chromosome 21 does not change in DS patients. The data is consistent a previous report that only 27% genes on chromosome 21 are differentially expressed in DS patientsGO enrichment analysis showed that the processes involved in basal metabolism, cell cycle, cell proliferation, and cell differentiation are affected by APP overexpression. Pathway analysis by IPA further supports the results of GO enrichment analysis, e.g. P53 signaling pathway which is involved in both cell cycle and cell proliferation. To further dissect the function of dysregulated genes, GO enrichment analysis was performed on upregulated and downregulated genes separately. We found that the upregulated genes are significantly involved in cell death, apoptosis and cell adhesion, which not only supports previous observations that APP overexpression promotes apoptosis and cell adhesion but also provides potential molecular mechanisms underlying these effects. Moreover, the upregulated genes are involved in negative regulation of transcription, translation, cell cycle and cell proliferation, while the downregulated genes are involved in transcription, translation, cell cycle process and cell proliferation. It highly suggests that APP overexpression may reduce cell basal metabolism and inhibit cell proliferation via upregulating or downregulating genes involved in aforementioned processes. Moreover, we demonstrated that the downregulation of PMSA5 and PMSB7 involved in cell cycle process, which may contribute to APP overexpression-induced proliferation impairment. Our data indicate that increased APP may contribute to the growth retardation and developmental delay in DS by inhibiting basal metabolism and cell proliferation.Human embryonic kidney (HEK) 293 cells and stable cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 1% L-glutamine and 1% penicillin/streptomycin (Invitrogen). HAW cells are HEK293 cells stably overexpressing wildtype APP. Transfections were performed with Lipofectamine 2000 (Invitrogen). Scramble siRNA and pre-designed siRNA of PSMA5 or PSMB7 were purchased from Applied Biosystems.RNA was isolated from cells using TRI-Reagent (Sigma-Aldrich). Thermoscript Reverse Transcription kit (Invitrogen) was used to synthesize the first strand cDNA following the manufacturer’s instruction. cRNA was amplified and purified by using IlluminaTotalPrep RNA amplification kit (Life Technologies). 1.5 μg cRNA of each sample was used for whole-genome gene expression direct hybridization assay with humanHT-12 v4 Expression Beadchip (Illumina) following the manufacturer’s instructions. Real-time PCR was performed following the Taqman One-Step RT-PCR protocol using premade primers and probes for each gene (Applied Biosystems). Amplification and detection were performed with the Smart cycler II (Cepheid). Three independent samples were assayed for each group.Cells were lysed in RIPA-Doc lysis buffer  supplemented with protease inhibitors (Boehringer Mannheim). 50 μg or 100 μg of total protein were resolved on 10–12% Tris-Glycine SDS-PAGE or 16% tris-tricine SDS-PAGE and transfered to polyvindylidinefluoride (PVDF-FL) membranes followed by immunoblotting. Rabbit anti-APP antibody C20 was used to detect APP and its C-terminal fragments (CTFs)5 cells were seeded on 35 mm dishes and the cell number was counted every three days. To monitor the proliferation ability, the cells were incubated with 10uM BrdU for 6 hours. After incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde for 20 minutes at room temperature. 2 M HCl was applied for 30 minutes at 37 °C followed by neutralization for 30 minutes with 0.1 M sodium borate. After permeabilization with 0.2% Triton solution for 15 minutes at room temperature, cells were blocked with goat serum followed by 2-hour incubation with goat anti-BrdU antibody (Sigma). Cells were washed with PBS and incubated with Cy3-conjugated anti-goat IgG (Thermo Scientific) for 1 hour. After washed with PBS, cells were stained with 4, 6-diamidino-2-phenylindole (DAPI) (Sigma). Cell images were taken by fluorescent microscope .To monitor the growth rate of HAW cells, 2.5 × 10t test was performed for the quantification of immunoblotting and BrdU labeling. Values of P   < 0.05 were considered significant. Differential gene expression, biological processes, canonical pathways and networks were analysed by Beadstudio, DAVID and Ingenuity Pathway Analysis software.Student’s How to cite this article: Wu, Y. et al. Regulation of global gene expression and cell proliferation by APP. Sci. Rep.6, 22460; doi: 10.1038/srep22460 (2016).


The morphological and physiochemical structure of the synthesized solar photocatalyst was investigated by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), energy-dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS). FESEM showed a mixture of nanowires and aggregated nanoparticles. This Al2O3 doped Mn3O4 nanomaterial exhibited high solar photocatalytic degradation in a short time when applied to brilliant cresyl blue (BCB). Thus, the synthesized nanoparticles can be used as an efficient solar photocatalyst for the degradation of BCB.Al Recent industrial development has improved living standards but due to insufficient environmental monitoring, the continuous discharge of massive industrial pollutants  is creating serious environmental problems. Since these pollutants are carcinogenic at trace levels for aquatic and non-aquatic organisms, a wide range of techniques including physical, chemical, and biological methods have been developed for water treatment –5. EnvirIn particular, the photocatalytic oxidation process using heterogeneous photocatalysis is regarded as a promising technology to decompose harmful pollutants into final non-toxic products –8. Howev2. Further studies reported the photocatalytic degradation of methyl orange by zinc ferrite-doped titania. Semiconductor iron (II) oxide has also been researched in photocatalytic bleaching of dyes. Furthermore, a large number of other systems such as transition metal-doped TiO2 and nitrogen-doped TiO2 have been utilized for the photosensitization of dyes [Nanostructured transition metal oxides have been considered important materials because of their electronic, optical, photonic, and catalytic properties. Their size reduction to nanoscale can effectively change their physical and chemical properties and specifically improve their potential . Since t of dyes .Manganese oxides have been extensively studied as a well-known transition metal oxide because of their unique chemical and physical properties. Manganese oxides have potential application in ion sieves, molecular sieves, catalysis, cathode materials for secondary rechargeable batteries, super capacitors, and new magnetic materials. Research has been reported into manganese oxide nanomaterials with different morphologies, such as nanorods, nanowires, nanotubes, and urchin-like nanostructure .Manganese oxides have been used as one of the most promising electrode materials for super capacitor applications with respect to their natural abundance, low cost, environmentally friendly nature, wide voltage window, and high specific capacitance . More sp2O3 doped Mn3O4 nanomaterial (NM) and characterized it by FESEM, EDS, XRD, XPS, FT-IR, and UV-visible. Further, we evaluated the photocatalytic performance of ΝΜ at different pH under solar light, using brilliant cresyl blue (BCB) as organic pollutant.In the research reported in this article, we synthesized Al3)3 · 9H2O), manganese nitrate tetrahydrate (Mn(NO3)2 · 4H2O) , BCB, sodium hydroxide NaOH, and 99 % pure ethanol were purchased from Sigma-Aldrich.Analytical grade chemicals including aluminum nitrate nonahydrate (Al(NOAluminum nitrate nonahydrate (3.7526 g) and manganese nitrate tetrahydrate (7.5634 g) were completely dissolved in 100.0 mL of distilled water, and a homogeneous solution at ambient temperature was obtained. The pH of the solution was adjusted to 10.50 with 0.2 M NaOH solution by drop-wise addition and constant vigorous stirring. Overnight, the solution was heated at 60–70 °C with constant stirring. The solution was then cooled to ambient temperature and the precipitate centrifuged at 2000 rpm. The supernatant solution was discarded and the precipitate preserved. The precipitate was washed with ethanol 1–2 times then allowed to dry at ambient temperature or in oven at 50–60 °C. The precipitate was ground and stored in clean, dry, and inert plastic vials.2O3 doped Mn3O4 nanomaterial may be elucidated on the basis of the following chemical reaction pathway:The growth of Al−1 on a Perkin Elmer (spectrum 100) FT-IR spectrometer. UV spectrum was recorded from 200 to 900 nm using UV-visible spectrophotometer . An XPS survey scan was made by Thermo Scientific K-Alpha KA1066 spectrometer (Germany) in the range of 0–1350 eV.The surface morphology of the nanoparticles was studied using a EOL scanning electron microscope . Elemental analysis was carried out using EDS (Oxford). X-ray diffraction patterns (XRD) were taken with a computer-controlled X’Pert Explorer, PANalytical diffractometer. FT-IR spectra were recorded in the range of 400–4000 cm2O3 doped Mn3O4 multi-metal oxide nanoparticles was evaluated through degradation of BCB under visible light irradiation. The dye is stable under visible light irradiation in the absence of photocatalyst.The photocatalytic activity of Al−4 M of BCB dye solutions were taken in different beakers and adjusted to pH 5 and 10, respectively, by drop-wise addition of 0.2 M NaOH solution under vigorous stirring. Then, 0.1193 and 0.1132 g of catalyst was added to the reaction solutions and kept in the dark, for physical adsorption of dye on catalyst surface. It has been reported in previous photocatalytic studies on TiO2 that besides the light absorption capability and charge transportation, the adsorption of reactant is also a critical factor [In photocatalytic degradation, two different 100.0 mL, 1 × 10l factor . The samλmax = 595.0 nm by spectrophotometer (LABOMED INC.). Absorbances were measured at time intervals 0, 10, 20, 40, 60, 90, 120, 140, 160, 180, 200, 220, 240, 260, 280, and 300 min. The controlled experiments were also performed under visible light without catalyst to measure any possible direct photocatalysis of dye.The dye solution of about 4–5 mL was pipetted out at regular intervals and the absorbance measured at 3O4 are shown in Fig. 3O4 displayed peaks related to Mn and O  indicated the presence of α-Al2O3 [3O4 phase. The peaks obtained for Mn3O4 are in good agreement with the literature  [Crystal phase and crystallinity of ΝΜ was checked by XRD, which is shown in Fig.  α-Al2O3 , 11. The24-0734) , 13.Fig.2O3 doped Mn3O4 nanomaterial was carried out by X-ray photoelectron spectroscopy. Figure 3/2 and Mn 2p1/2 has binding energy (BE) values of 644.26 and 655.10 eV, respectively  values of 644.10, 655.07, and 531.04 eV, respectively 2 vs. hv  solution was carried out in the presence of the photocatalyst under visible light irradiation. Metal oxide showed efficient activity for degradation of BCB dye at different pH under solar light irradiation Fig. . FurtherThe Langmuir–Hinshelwood (L-H) model was successfully applied to the visible light photocatalytic degradation of organic dye and represented the best linear relationship between photocatalytic degradation rate and initial concentration of organic pollutant . The ratr is the degradation rate of dyes (organic pollutant), Kr is the reaction rate constant, K is the equilibrium constant, and C is the reactant concentration. When C was very small, KC was negligible, so that Eq. t = 0, C = Co), it became Eq. where Co was the initial concentration of dye, and C was the concentration at time “t”. Using Eq. C/Co), corresponding to ln(A/Ao) vs. irradiation time.C/Co) as a function of irradiation time showed linearity. Table Kapp, evaluated from the slopes of the linear plot, t1/2 parameters  and regression relative coefficient values.The apparent rate constant of photocatalytic activity was independent of adsorption and the concentration of the dye remaining in the solution. Figure 2•−) (Eq. 2O2) followed by hydroxyl radical (•OH) (Eq. +) in VB generates hydroxyl radicals (•OH) from water solvent  Eq. , since t•OH) Eq. . Furthervent Eq. .Fig. 9Sc2•− and •OH radicals on pollutant species (organic dye) lead to the degradation (oxidation) of the dye molecule  and hydroxyl (•OH) radicals generated by oxidation and reduction reaction of O2 and H2O, respectively, are primarily responsible for photocatalyzing the degradation of organic pollutant BCB under the irradiation of solar light. The decay of BCB follows the pseudo first-order kinetics which satisfied the Langmuir-Hinshelwood (L-H) kinetic model. This research represents an important advance in the synthesis of novel co-doped oxides for the photocatalytic degradation of organic pollutant under visible light irradiation for industrial effluent.A simple low-temperature and low-cost solar photocatalyst based on Al


Astaxanthin, a xanthophyll carotenoid found in aquatic animals, is known to possess antioxidant, anti-inflammatory, and antineoplastic properties. The present study examined the effects of astaxanthin on the development of azoxymethane (AOM)-induced colonic premalignant lesions in C57BL/KsJ-db/db mice were administered 4 weekly subcutaneous injections of AOM (15 mg/kg body weight) from 5 weeks of age and subsequently, from 1 week after the last injection of AOM, were fed a diet containing 200 ppm astaxanthin throughout the experiment (8 weeks).Male GPx1, SOD1, and CAT in the colonic mucosa of AOM-treated db/db mice. The expression levels of IL-1β, IL-6, F4/80, CCL2, and CXCL2 mRNA in the colonic mucosa of AOM-treated mice were significantly decreased by astaxanthin. Dietary feeding with astaxanthin also resulted in a reduction in the numbers of NF-κB- and PCNA-positive cells that were increased by AOM exposure, in the colonic epithelium.The development of colonic premalignant lesions, i.e., aberrant crypt foci and β-catenin accumulated crypts, was significantly inhibited in mice treated with astaxanthin than in mice fed the basal diet. Astaxanthin administration markedly reduced urinary levels of 8-OHdG and serum levels of d-ROMs, which are oxidative stress markers, while increasing the expression of mRNA for the antioxidant enzymes These findings suggest that astaxanthin inhibits the development of colonic premalignant lesions in an obesity-related colorectal carcinogenesis model by reducing oxidative stress, attenuating chronic inflammation, and inhibiting NF-κB activation and cell proliferation in the colonic mucosa. Astaxanthin, therefore, may be a potential candidate as a chemoprevention agent against colorectal carcinogenesis in obese individuals. Obesity, a growing health concern worldwide, is a result of excess energy intake and insufficient exercise. Obesity is associated with increased risk of diseases with high mortality, such as ischemic heart disease, stroke, and cancer . In partSeveral pathophysiological mechanisms linking obesity and the development of CRC have been elucidated, including the emergence of insulin resistance, imbalance of adipokines, induction of oxidative stress, and a state of chronic inflammation -5. ObeseAstaxanthin, a conventional red-colored xanthophyll, is an oxygenated carotenoid derivative occurring naturally in a wide variety of living organisms including microalgae, fungi, salmon, trout, shrimp, and some birds ,15. Astadb/db (db/db) mice, which lack the long form of the leptin receptor, develop hyperphagic obesity and diabetes [db/db mice [db/db mice [C57BL/KsJ-diabetes . Interes/db mice . A precl/db mice  has prov/db mice ,12,23-25/db mice -28, usindb/db mice were purchased from Japan SLC, Inc.  and were maintained humanely at the Gifu University Life Science Research Center in accordance with the Institutional Animal Care Guidelines. AOM was purchased from Sigma-Aldrich . Astaxanthin from Haematococcus pluvialiswas was supplied by Fuji Chemical Industry .Four-week-old male db/db mice were divided into the following 4 groups: untreated control (n = 10); 200 ppm astaxanthin alone (n = 10); AOM alone (n = 10); and, AOM and 200 ppm astaxanthin (n = 10). At 5 weeks of age, mice in AOM alone group and AOM + astaxanthin group were injected with AOM (15 mg/kg body weight) subcutaneously once a week for 4 weeks. None treatment group and AOM alone group were fed the basal diet CRF-1  throughout the experiment. The composition of the CRF-1 diet was as follows: 8.1 g/100 g water, 22.6 g/100 g protein, 5.6 g/100 g fat, 6.6 g/100 g minerals, 3.3 g/100 g fiber, and 53.8 g/100 g carbohydrates. Astaxanthin alone group and AOM + astaxanthin group were fed the basal diet supplemented with 200 ppm astaxanthin for 8 weeks, starting 1 week after the last injection of AOM. The dosage of astaxanthin was determined according to a previous report [All experimental protocols involving animals were approved by the Animal Research Committee, Gifu University Graduate School of Medicine. Forty male s report . Food in2/colon) were then resected and embedded in paraffin, and a total of 20 serial sections (each 4 μm thick) per mouse were cut by an en face preparation to identify BCAC intramucosal lesions [The numbers of ACF and BCAC were determined according to standard procedures ,23,24,29 lesions ,23,24.1-to-S phase marker, and for phospho-nuclear factor-κB (NF-κB) p65 were run on histological sections to estimate cell proliferative activity and NF-κB activity, respectively, in the colonic crypts [Immunohistochemistry for β-catenin was performed using the labeled streptavidin-biotin method  to count the number of BCAC ,23,24. Tc crypts ,23, usinc crypts ,23.tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, F4/80, chemokine (C-C motif) ligand (CCL)2, chemokine (C-X-C motif) ligand (CXCL)2, glutathione peroxidase (GPx)1, superoxide dismutase (SOD)1, catalase (CAT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were obtained from Primer-BLAST . The cDNA was synthesized from 0.2 μg of total RNA using the High Capacity cDNA Reverse Transcription Kit . A quantitative real-time reverse transcription-PCR (RT-PCR) analysis was performed using a LightCycler Nano  with FastStart Essential DNA Green Master (Roche Diagnostics). The PCR cycling conditions were 95°C for 10 min, followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 15 s. The sequences of specific primers amplifying Blood samples were collected from the inferior vena cava at sacrifice after 6 hours of fasting for chemical analyses. The serum concentrations of insulin , glucose , adiponectin , leptin (R&D Systems), triglyceride , and TNF-α (R&D Systems) were determined using an enzyme immunoassay, according to the manufacturer’s protocol.Urine 8-hydroxy-2’-deoxyguanosine (8-OHdG) levels were measured using an enzyme-linked immunosorbent assay kit . Serum levels of hydroperoxide, a marker for oxidative stress, were determined using the derivatives of reactive oxygen metabolites (d-ROMs) test  .P value was less than 0.05.The measures are presented as mean ± SD and were statistically analyzed using the GraphPad InStat software program, Version 3.05  for Macintosh. One-way analysis of variance (ANOVA) was used to compare groups. If the ANOVA analysis indicated significant differences, the Tukey–Kramer multiple comparisons test was performed to compare the mean values among the groups. The differences were considered significant when the two-sided P < 0.05) at the termination of the experiment. This might be due to the toxicity of AOM, as observed in our previous studies [P < 0.05). No significant differences were observed in the mean relative weights of adipose tissue among the groups. Histopathological examination of the liver, kidney, and spleen confirmed the absence of toxicity of dietary astaxanthin (data not shown).During the experiment, dietary feeding with astaxanthin did not cause any clinical symptoms. As listed in Table  studies ,23,24. TP < 0.05). Moreover, the number of BCAC , indicating that two types of precursor lesions for CRC were suppressed by astaxanthin administration. These findings suggest that astaxanthin prevented the early phase of obesity-related colorectal carcinogenesis.ACF Figure A developC Figure B, which P < 0.05), which reflects DNA damage induced by oxidative stress, and serum d-ROMs , marker for hydroperoxide levels of serum, as compared to those observed in the control group. However, the level of 8-OHdG significantly decreased with astaxanthin administration . As shown in Figure GPx1 mRNA, which encodes an antioxidant enzyme, was reduced by AOM injection (P < 0.05), but astaxanthin significantly increased the expression of GPx1, as well as of SOD1 and CAT, both of which encode antioxidant enzymes, in the colonic mucosa of db/db mice treated with AOM (P < 0.05).Oxidative stress is implicated in obesity-related colorectal tumorigenesis . TherefoP < 0.05), but not in the astaxanthin-treated mice, than those in the control mice. As shown in Figure TNF-α, IL-1β, IL-6, and F4/80 (P < 0.05 compared to the untreated control group) in the colonic mucosa of group treated with AOM alone. However, astaxanthin administration significantly decreased the expression of IL-1β, IL-6, and F4/80 mRNA in the colonic mucosa of the AOM-treated db/db mice (P < 0.05). The expression of CCL2 and CXCL2, which are associated with colorectal carcinogenesis [db/db mice was also significantly decreased by astaxanthin treatment (P < 0.05).Chronic inflammation plays a critical role in the pathogenesis of obesity and CRC development ,31. Therogenesis -34, in tP < 0.001 for each comparison). As shown in Figure P < 0.01), which had been increased by AOM injection (P < 0.001). These findings indicate that astaxanthin significantly inhibits NF-κB activity and cell proliferation in the colonic mucosa of AOM-treated db/db mice.NF-κB activation is critically involved in the progression of inflammation and the activation of cell proliferation in colonic mucosa . TherefoP < 0.05). None of these metabolic parameters had been altered by astaxanthin administration at the end of this study.Diabetes, dyslipidemia, and adipokine imbalance, which are complications often associated with obesity, are involved in colorectal tumorigenesis -5. Theredb/db obese mice, mainly through the reduction of oxidative stress and attenuation of inflammation.Obesity, which is one of the most serious healthcare problems worldwide, is a significant risk factor for the development of CRC ,5. OxidaSaturated fatty acids from obesity-induced lipolysis are capable of activating macrophages and thereby activating NF-κB signaling, which in turn leads to transcriptional activation of genes encoding pro-inflammatory factors including IL-1β and IL-6 . IL-1β pGPx1, SOD1, and CAT mRNA, which encode antioxidant enzymes, in the colonic mucosa were increased by astaxanthin intake in AOM-injected db/db mice. These results strongly indicate that attenuation of oxidative stress and recuperation from high oxidation state via antioxidative effects are critical mechanisms by which astaxanthin suppressed the occurrence of premalignant lesions, ACF and BCAC, in obese mice. It should be mentioned that astaxanthin has even been called a super-antioxidant because it is a superior antioxidant and scavenger of free radicals as compared with other carotenoids such as β-carotene [Obesity and chronic inflammation are often accompanied with increased generation of reactive oxygen species (ROS), which are derivatives of molecular oxygen such as superoxide and hydrogen peroxide. These derivatives can induce mutagenic changes and may damage DNA repair proteins, resulting in cancer development ,42. Canccarotene .Epidemiologically, it is still unclear whether the intake of carotenoids such as astaxanthin is associated with a reduced risk of CRC development. The results of randomized trials using β-carotene supplementation provided no evidence to support an effect of carotenoids on CRC chemoprevention ,46. Rathdb/db mice by ameliorating insulin resistance and improving adipokine imbalance. However, abnormalities in serum levels of glucose, insulin, adiponectin, and leptin were not improved by astaxanthin administration in this study. We suggest that this was likely due to the duration of the experiment (8 weeks) and the particular animal model studied, because previous studies demonstrating effects of astaxanthin on insulin sensitivity were long-term studies (22 weeks) [db/db mice, in which insulin resistance was not improved by astaxanthin, but apoptosis of retinal ganglion cells was attenuated via the suppression of oxidative stress [Previous reports using metabolic syndrome animal models have shown that astaxanthin reduces insulin resistance, recovers insulin sensitivity, and increases serum levels of adiponectin ,52. A do2 weeks)  and the 2 weeks) ,52. Our e stress . Future In summary, the results from this study showed that reduction of oxidative stress and attenuation of inflammation in the colonic mucosa are crucial mechanisms by which astaxanthin acts to prevent the early phase of obesity-related colorectal carcinogenesis. Since the risk of CRC increases with obesity and obesity-related metabolic abnormalities, which are impending health crises worldwide, targeting obesity-related metabolic abnormalities, including chronic inflammation and induction of oxidative stress, may be an attractive and effective strategy for preventing the development of CRC in obese individuals. Astaxanthin seems to be a potentially effective and viable candidate for this purpose because this agent attenuates chronic inflammation while reducing oxidative stress without causing side effects.


Mycobacterium avium subspecies paratuberculosis (MAP) with T1D in adult Sardinian patients. To enforce this finding, we investigated the presence of antibodies against ZnT8 and proinsulin (PI) with respective homologous epitopes: MAP3865c133–141/ZnT8186–194, MAP3865c125–133/ZnT8178–186, MAP2404c70–85/PI46–61, and MAP1,4αgbp157–173/PI64–80, in 23 children at risk for T1D, formerly involved in the TRIGR study, and 22 healthy controls (HCs). Positivity to anti-MAP and homologous human peptides was detected in 48% of at-risk subjects compared to 5,85% HCs, preceding appearance of islet autoantibodies. Being MAP easily transmitted to humans with infected cow's milk and detected in retail infant formulas, MAP epitopes could be present in extensively hydrolyzed formula and act as antigens stimulating β-cell autoimmunity.As numerous studies put in evidence the increasing incidence of type 1 diabetes (T1D) in children, an early diagnosis is of great importance to define correct treatment and diet. Currently, the identification of classical islet autoantibodies is the primary biomarker for diagnosis in subjects at risk, especially in pediatric patients. Recent studies suggest that detection of antibodies against ZnT8 protein in preclinical phase can predict the development of T1D. We previously demonstrated a significant association of This process starts in early infancy and can occur over many years during childhood without developing clinical symptoms. The primary biomarkers for diagnosis in subjects at risk involve identification of Abs to islet cell antigens (ICA), glutamic acid decarboxylase 65 (GADA), tyrosine phosphatase-related insulinoma-associated 2 molecule (IA-2), and insulin (IAA), among which anti-(pro)insulin Abs appear at a younger mean age [ Mycobacterium avium subspecies paratuberculosis (MAP), a bacterium putatively associated with T1D [Type 1 diabetes (T1D) is an autoimmune disease with increasing incidence in youth worldwide [mean age  and are mean age . Recentlwith T1D .β-cell immunity [MAP is the causative agent of Johne's disease in livestock ruminant species and has been associated with Crohn's disease in humans , 12. As immunity –26, the immunity . The tri Helicobacter pylori (HP) were assessed in order to achieve a picture of seroreactivity to a pathogen not associated with T1D and a possible cross-reaction with anti-MAP Abs.Following our previous outcomes hypothesizing a possible role of MAP as an environmental agent in T1D onset , 10, in n = 22, mean age 4,3 ± 2,1 years) were provided by the Tor Vergata University of Rome based on a single collection scheme. Blood samples were collected after obtaining written informed consent for all study participants from a parent or caretaker and plasma was separated by sedimentation. The study protocols were approved by the Ethical Committees of the University of Sassari, the St. Michele Hospital of Cagliari, and the Tor Vergata University of Rome, Italy, in accordance with the Declaration of Helsinki.In the framework of TRIGR study, 23 Sardinian children at risk of T1D  attending the Department of Diabetes, St. Michele Hospital of Cagliari, Italy, were enrolled in the study and followed in time in order to identify a correlation between the presence of Abs and the onset of diabetes. The first blood samples were collected at birth with further annual collections up to 10 years, giving in total 139 samples over the period of 2002–2012 with the mean age 6,65 ± 2,74 years at the end of follow-up. In addition, all at-risk subjects (ARS) were analyzed for the presence of ICA, GADA, IA-2, and IAA autoantibodies. We analyzed relative plasma samples for Abs against ZnT8, PI, and homologous MAP peptides. Reference control samples of age-matched healthy volunteers  were determined for all ARS as described elsewhere .133–141 (LAANFVVAL) and MAP3865c125–133 (MIAVALAGL), a hypothetical protein MAP2404c70–85 (RGFVVLPVTRRDVTDV), and a glucan branching protein MAP1,4αgbp157–173 (GTVELLGGPLAHPFQPL) along with their respective homologs ZnT8186–194 (VAANIVLTV), ZnT8178–186 (MIIVSSCAV), PI46–61 (RGFFYTPKTRREAEDL), and PI64–80 (GQVELGGGPGAGSLQPL) [Formerly identified immunodominant MAP-derived peptides of a cation transporter MAP3865cAGSLQPL)  were syn1–11 (MIIGGGVSGCA) derived from HP quinone oxidoreductase J0I929_HELPX protein (UniProtKB accession number J0I929) and T2T4W3_HELPX99–105 (AGIVLTV) derived from HP preprotein translocase subunit T2T4W3_HELPX (UniProtKB accession number T2T4W3), respective homologs of ZnT8178–186 and ZnT8186–194, were synthesized by the same manufacturer at >90% purity. All purified peptides were stored at −80°C in single-use aliquots as 10 mM solutions.In addition, peptides J0I929_HELPXαgbp/PI, MAP2404c/PI, and HELPX/ZnT8 peptides were performed as described previously [1–11  and T2T4W3_HELPX99–105 . Interassay coefficient of variation (CV) for the 8 peptide-based ELISAs ranged from 6,7% to 8,4%, whereas the intra-assay CV accounted for 2,8% upon 20 Abs reactivity tests. GraphPad Prism ver. 6.0 software was used for statistical analyses of the assay with levels of significance determined through Student's t-test (95% CI) or the Mann-Whitney U test for not normally distributed data.Indirect ELISAs to detect Abs specific for MAP3865c/ZnT8, MAP1,4eviously . Data waαgbp157–173 (M1) Abs, compared to 9,09% of HCs. The same prevalence among ARS was detected for the homologous PI64–80 (P1) peptide, but only in 13,64% of HCs.In the present study population, 10 out of 23 ARS  were positive for anti-MAP1,470–85 (M2) were recognized by 7 ARS  and 13,64% HCs, while 6 ARS  and 9,09% of HCs were positive for its homolog PI46–61 (P2) (Abs against MAP2404c–61 (P2) .133–141 (M3) was registered for 6 ARS  and 9,09% of HCs, whereas 5 ARS  were positive for the homologous ZnT8186–194 (Z3) peptide, comparing to 4,55% among HCs.Reactivity to MAP3865c125–133 (M4) Abs were found in 5 ARS  and in none of the HCs. Recognition of its homolog ZnT8178–186 (Z4) among the analyzed subjects accounted for 6 ARS  and 4,55% of positivity for HCs. The frequencies of Abs here presented are noncumulative values based on evaluation whether positivity was detected or not during the follow-up.Anti-MAP3865cn = 11) reacted to any of the analyzed peptides compared to 22,73% of age-matched HCs. A high degree of correlation  was found between anti-MAP and the respective anti-PI/ZnT8 Abs titers , 3 for IAA and/or IA2 (13%), and 2 for GAD antibodies  . Overalls titers .133–141, MAP3865c125–133, MAP2404c70–85, and MAP1,4αgbp157–173, ZnT8186–194, ZnT8178–186, PI46–61, and PI64–80 ; two of them additionally recognized Abs against MAP2404c70–85/PI46–61 homologs and anti-MAP3865c125–133/ZnT8178–186 Abs were found in one child. Four children presented Abs positivity against nonhomologous peptides MAP3865c133–141, MAP3865c125–133, MAP2404c70–85, ZnT8186–194, and PI46–61. The overlap in reactivity against homologous peptides among positive HCs (n = 5) accounted for 40% and regarded only two subjects with Abs directed against at least 4 epitopes: one HC recognized MAP1,4αgbp157–173/PI64–80 and MAP2404c70–85/PI46–61 peptide pair while the other one showed positivity to MAP3865c133–141/ZnT8186–194 and MAP2404c70–85/PI46–61 homologs. Nonhomologous reactivity was detected for MAP1,4αgbp157–173, PI64–80, MAP3865c133–141, ZnT8178–186, and MAP2404c70–85.Interestingly, of the 23 ARS followed in time, 5 were positive for all the peptides MAP3865c PI64–80 ; in otheFive of the positive children presented autoimmunity against both the studied peptides and classical islet antibodies. Furthermore, positivity to our peptides not only appeared before detection of classical autoantibodies within 6 months of age , but was1–11 or T2T4W3_HELPX99–105, were detected in one of ARS  and the results were not statistically significant . No positive cases were observed among controls , a correlation with diet may reveal whether positivity to ZnT8, PI, and homologous MAP peptides in our samples is significantly associated with formula type.The present study has a few limitations. We avoided describing Sardinian control subjects as the relative data have been already published . In this


Background: Protein-disulfide isomerase (PDI) has previously been identified to bind bisphenol A (BPA), an endocrine disrupter.Results: BPA inhibited Ero1α-PDI-mediated disulfide bond formation.Conclusion: BPA significantly inhibited the Ero1α and PDI oxidative cycle, probably through closure of the substrate- and Ero1α-binding pocket in the PDI b′ domain.Significance: BPA may have inhibitory effects on oxidative folding of secretory and membrane proteins. Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b′ domain, preventing PDI from binding to unfolded proteins. The b′ domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b′ domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding β-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation. A) and accumulates in tissues once taken up by living organisms. In addition to its antagonist/agonist effects on steroid hormone receptors, BPA induces a number of detrimental effects on the central nervous system, including induction of abnormal behavior in mice and morphogenic malformation in Xenopus laevis . The a and a′ domains contain a catalytic active site composed of a CGHC sequence, whereas the central domains, b and b′, possess hydrophobic surfaces responsible for the binding of incompletely folded protein substrates  . PDI, anterminus , 10  catalyzed by Ero1α and PDI. BPA induced significant spatial rearrangement of the a and b′ domains of PDI, leading to a more compact overall structure. Thus, BPA probably inhibits the Ero1-PDI disulfide bond formation pathway of the ER, an essential organelle engaged in the synthesis of secretory and membrane proteins.We previously found that BPA binds the PDI y of PDI , 26. BecBPA was purchased from Wako Pure Chemical Industries, Ltd. . All other chemicals and solvents used were of the highest grade available.full-length, PDI(120–353), and PDI(354–509) fragments were constructed as described previously   .m phosphate buffer (pH 7.4) at room temperature, using a quartz cell with a 1.0-mm path length for far-UV (260–200 nm) and a 10-mm path-length for near-UV spectra (340–250 nm). Protein concentrations were as follows: for PDIfull-length, 0.95 mg/ml in near-UV spectra and 0.24 mg/ml in far-UV spectra; for PDI(120–353), 0.69 mg/ml in near-UV spectra and 0.31 mg/ml in far-UV spectra; for PDI(354–509), 0.93 mg/ml in near-UV spectra and 0.31 mg/ml in far-UV spectra. All samples were centrifuged at 15,000 × g for 15 min, and then supernatants were used for measurements.CD measurements were performed on a JASCO J-600 and J-720 spectropolarimeter . The CD spectra of PDI(120–353) and PDI(354–509) were measured in 20 m−1, 120 °C h−1, and 200 °C h−1 for PDI(120–353) and 200 °C h−1 for PDI(354–509). Protein solutions were dialyzed against 20 mm phosphate buffer (pH 7.0) and thereafter centrifuged at 15,000 × g for 15 min. Supernatants were filtered through a 0.22-μm pore size membrane before measurement. Protein concentration of PDI(120–353) was 0.32 mg/ml for 200 °C h−1 and 0.97 mg/ml for 60 and 120 °C h−1. Protein concentration of PDI(354–509) was 0.32 mg/ml. Reversibility of heat denaturation for PDI(120–353) was performed at a scan rate of 60 °C h−1 in 20 mm phosphate buffer (pH 7.0) with a protein concentration of 0.72 mg/ml. Thermograms were integrated using ORIGIN software  to estimate binding constants.DSC measurements were performed using a Microcal VP-capillary DSC platform  at a scan rate of 60 °C hg for 10 min, and the supernatants were filtered through a 0.22-μm pore size membrane before measurement. The protein concentration was 0.89 mg/ml for PDI(120–353) and 1.18 mg/ml for PDI(354–509).The hydrodynamic sizes of PDI(120–353) and PDI(354–509) in solution were measured with a Zetasizer NanoZS , based on the principle of dynamic light scattering . BrieflyTo predict the structure of PDI(120–353) and PDI(354–509), a protein homology/analogy recognition engine (Phyre) was used .m phosphate buffer (pH 7.0), and SAXS profiles for PDI(120–353) in the presence or absence of BPA (with a 1:1 molar ratio of PDI(120–353)/BPA) were collected in a concentration range of 1.5–4.0 mg/ml. SAXS profiles for bovine serum albumin  were also collected as a reference for determining the relative molecular weight of PDI(120–353). All samples were centrifuged at 15,000 × g for 15 min, and supernatants were used for measurements.SAXS data were collected on the BL45XU beamline at the RIKEN SPring-8 Center  . The x-rI(0) and gR are the forward scattering intensity at Q = 0 and the radius of gyration, respectively (I(0) value is proportional to the averaged molecular weight and concentration. The pair distribution function, P(r), was calculated by indirect Fourier transformation using GNOM , reduced glutathione (300 μm), human PDI (10 μm), the hyperactive form, including mutations of C104A and C131A, of human Ero1α (4 μm) (m). The oxidation of NADPH by glutathione reductase led to a decrease in absorbance at 340 nm (A340). A molar extinction coefficient of 6,200 m−1 cm−1 for NADPH was used for the calculations. The reaction was initiated by adding Ero1α. Changes in absorbance at 340 nm were monitored with a Hitachi U-3310 spectrophotometer at 30 °C in 50 mm Tris-HCl, pH 7.5, 0.3 m NaCl. IC50 values were calculated by curve fitting using ImageJ.Glutathione reductase (1 unit) was incubated with NADPH (200 μα (4 μm) , and varm) was refolded at 30 °C in oxygen-saturated buffer containing 50 mm Tris-HCl (pH 7.5), 300 mm NaCl, 1 μm PDI, a 1 μm concentration of the hyperactive form of Ero1α, and 40 μm BPA. For the Prx4-PDI- or glutathione-PDI-mediated folding of BPTI, the same procedure was used, but the buffer was degassed by flushing N2 gas. Reactions were performed at 30 °C in buffer containing 50 mm Tris-HCl (pH 7.5), 300 mm NaCl, 1 μm PDI, 0.1 μm Prx4, 200 μm H2O2, and 40 μm BPA (m Tris-HCl (pH 7.5), 300 mm NaCl, 1 μm PDI, 2 mm GSH, 0.2 mm GSSG, and 40 μm BPA (Reduction and denaturation of BPTI (Takara Bio Inc.) were performed as described previously , 35–37. 35–0 μm BPA , 28 or b0 μm BPA , 39. Them in buffer containing 40 mm HEPES-KOH, pH 7.5, PDI (4 μm) and BPA (20 or 40 μm) under constant stirring and incubated at 45 °C during the measurement. Thermal aggregation was monitored by measuring light scattering using a Hitachi Spectrofluorometer F2500. The wavelength of light entered and monitored was 350 nm.The chaperone activity of PDI was measured using luciferase as a model substrate . To moni2. Where indicated, cells were treated with 10 mm dithiothreitol (DTT), 1 mm diamide, or the indicated concentrations of BPA for the indicated times before preparing samples. To analyze the redox state of PDI in cells, cells were washed twice with phosphate-buffered saline (PBS) and treated with ice-cold 10% trichloroacetic acid. After a 20-min incubation on ice, cellular proteins were collected by centrifugation, washed once with ice-cold acetone to remove acid, and dissolved in alkylation buffer (100 mm Tris-HCl (pH 6.8), 2% SDS) containing 5.5 mm PEG-maleimide 2000  and protease inhibitors  (HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% fetal bovine serum at 37 °C under 5% COluoride) . The samluoride)  and withm DTT for 10 min to reduce PDI (pulse)  . DTT wasA340 due to the consumption of NADPH . BPA markedly inhibited Ero1α-mediated catalysis of PDI oxidation in a dose-dependent manner, with an IC50 of 41 μm . To examine whether BPA acts on PDI or Ero1α in exerting this inhibitory effect, we analyzed the binding of BPA to PDI or Ero1α by surface plasmon resonance (SPR). We observed that BPA directly associated with PDI but not with Ero1α (D). Hiroi et al. . It is conceivable that BPA inhibits Ero1α-mediated catalysis of PDI oxidation by binding a site on PDI critical for functional interplay with Ero1α. We next investigated the effect of BPA on the chaperone activity of PDI using luciferase as a model substrate (E). Although the presence of PDI prevented the luciferase aggregation to a significant extent, the addition of BPA again caused the aggregation in a dose-dependent manner. Thus, BPA has an inhibitory effect on the chaperone activity of PDI.To determine the rate of Ero1α-mediated PDI oxidation, we previously developed an NADPH consumption assay, in which oxidation of NADPH is coupled to Ero1α-mediated catalysis of PDI oxidation through the intermediary actions of glutathione reductase and glutathione . In thisof NADPH C, left. of 41 μm C, right.th Ero1α D. Hiroi i et al.  indicateon assay C. It is ubstrate E. Althou5–Cys55, Cys14–Cys38, and Cys30–Cys51). Glutathione-mediated oxidative folding pathways of BPTI have been well characterized by HPLC (A) (B and C). Notably, whereas BPA inhibited both the early and late steps of BPTI folding, the effect was even more pronounced in the conversion of the des species to the N species than in the initial disulfide introduction during the early stage of folding. Previous in depth studies indicate that PDI plays a critical role in the formation of native disulfide bonds within partially structured and kinetically trapped folding intermediates, especially des species, of BPTI during the late steps of folding  , B and C folding , 28. Lik folding .A). In line with this, the early stage of BPTI folding catalyzed by Prx4-PDI pathway was little affected by the addition of BPA (B). However, the late steps of BPTI folding catalyzed by this pathway were significantly inhibited (C), as was the case with glutathione-PDI-mediated folding (B). We thus infer that BPA compromises the ability of PDI to recognize folding intermediates as well as the functional interplay between PDI and Ero1α.Meanwhile, BPA had no effect on the Prx4-catalyzed PDI oxidation A. In linn of BPA B. Howevenhibited C, as was folding B. We thuin vitro, we envisaged that BPA might slow down the reoxidation of PDI in cells. To test this possibility, we first treated the cultures of HeLa cells with 10 mm DTT for 10 min to reduce PDI and washed the cells to remove DTT from the culture (Because BPA inhibited the ability of Ero1α to oxidize PDI  culture . We thenA). In addition to the oxidized and reduced forms of PDI, we detected a number of bands with different apparent molecular masses that reacted with antibody to PDI . We suggest that the latter bands represent disulfide-linked complexes between PDI and its partners for two reasons. First, they ran slower than the monomeric forms of PDI. Second, when the samples were treated with reducing agent before electrophoresis, these bands disappeared, giving rise to bands corresponding to the monomeric forms of this protein . Remarkably, the treatment of the culture with BPA caused the accumulation of the reduced form of PDI with concomitant decreases in the levels of the disulfide-linked complexes . This effect was enhanced as the BPA concentration increased, suggesting that BPA slowed down the reoxidation of PDI in the cells.To determine the oxidation (disulfide-bonded) status of PDI in the cells, we treated the cells directly with acid to prevent post-harvest oxidation of cysteines and then alkylated free cysteines with PEG-maleimide 2000 . This modification stabilizes disulfide-linked complexes and retards the migration of the reduced form of proteins on a gel . The oxiFig. 5B). As observed clearly in Fig. 5B, lanes 3–6, BPA treatment increased the level of alkylation on PDI in a dose-dependent manner, indicating again that BPA can slow down the reoxidation of PDI in the cells.Because disulfide bonds that form within a protein or between proteins can alter the mobility of the protein on a gel, we treated the cell lysates with a reducing agent to observe the oxidation status of PDI itself more directly. Indeed, separation of the PEG-maleimide-treated cell lysate on a reducing gel allowed us to identify five distinct bands whose changes in the mobility should reflect changes in the numbers of alkylation on free cysteines in PDI B. As obsm BPA for various times and looked at the oxidation status of PDI in the cells. As observed in C and D, BPA caused the reduction of PDI in HeLa cells in a time-dependent manner, indicating that BPA can have a great impact on the redox homeostasis of PDI in the cells. These findings are consistent with the observation that BPA can inhibit the ability of Ero1α to oxidize PDI in vitro (see above).We next proceeded to examine whether BPA has an impact on the oxidation status of PDI in the cells. For this purpose, we cultured HeLa cells without DTT treatment in the presence of 400 μA) as well as toward PDI (A). In line with this, the addition of BPA had little effect on the HPLC profiles, indicating the time course of BPTI oxidative folding (B).To further examine the possible role of BPA as an inhibitor of oxidative pathways other than Ero1α-PDI, we analyzed Prx4-catalyzed ERp46 oxidation in the presence of BPA. A BPA titration assay showed little or no effect on Prx4 oxidative activity toward ERp46 A as wellward PDI A. In lin folding B.50 value was 538 μm, more than 10-fold higher than that of Ero1α-mediated PDI oxidation in the presence of BPA (IC50 = 41 μm) (C). Thus, BPA had the ability to more or less inhibit the oxidative pathways constituted by Ero1α and PDI family members .By contrast, BPA had weak but significant inhibitory effect on Ero1α-mediated ERp46 oxidation. The IC= 41 μm) C. Thus, A, left), a slight but significant difference was observed in the near-UV region; a CD signal at ∼290 nm increased upon titration of BPA . The results suggest that although BPA had little effect on secondary structure, BPA induced a slight but significant change in the chemical environment around the tryptophan residues of PDI.The functional defects caused by BPA suggest that BPA may induce conformational changes in PDI. To examine this possibility, we measured near-UV and far-UV CD spectra of PDI in the presence or absence of BPA. Although there was no significant change in the far-UV region A, left, n of BPA A, right.a and intact domains b and b′ , and that of PDI(354–509), which consists of the x-linker and domains a′ and c . The former fragment contains only one tryptophan residue (Trp130) in the C-terminal region, whereas the latter contains one tryptophan residue in the x-linker region and two in domain a′.To explore which region of PDI undergoes BPA-induced conformational changes, we measured near-UV CD spectra of PDI(120–353), which consists of the C-terminal helix of domain b and b′ B, left, a′ and c B, right.C), indicating that the secondary and tertiary structure of PDI(354–509) were unaltered upon the addition of BPA. By contrast, PDI(120–353) exhibited a marked increase in CD signal at ∼290 nm in proportion to the concentration of BPA added , whereas far-UV CD spectra were insensitive to BPA . These results strongly suggest that the location and surrounding environment of Trp130 contained in domain a of PDI(120–353) were greatly affected by BPA despite the lack of large conformational changes in secondary structure.CD spectra of PDI(354–509) in the near- and far-UV regions were almost identical regardless of the presence or absence of BPA C, indicaPA added D, right,e to BPA D, left. A shows that the DSC curves for PDI(120–353) were shifted to higher melting temperature (mT) values in accordance with the BPA/PDI(120–353) molar ratio; the mT of PDI(120–353) increased by 4.4 °C in the presence of a 10-molar equivalent excess of BPA at a scan rate of 60 °C h−1 . Precise analysis of thermal denaturation requires confirmation of its reversibility. To this end, a second run was performed by reducing the temperature after the first run. Successive rescans of PDI(120–353) showed a superimposable curve (89.3%) at a scan rate of 60 °C h−1 at pH 7.0 , indicating that the observed unfolding transition was highly reversible. Changes in unfolding enthalpy by BPA binding are listed in To investigate possible changes in the thermal stability of PDI(120–353) upon the addition of BPA, we performed DSC analysis. Fig. 80 °C h−1 . SimilarmT and T0 are melting temperatures in the presence and the absence of BPA, respectively, [L]Tm is the concentration of unbound BPA at mT, n is the difference in the number of bound molecules of BPA in the unfolded and folded states, K is a binding constant, R is the gas constant, and ΔpC is a heat capacity change due to heat denaturation of a protein. The ΔpC value upon PDI(120–353)-BPA complex formation was estimated to be 1.66 × 10 kJ mol−1 K−1, using the water-accessible nonpolar surface area and polar surface area of PDI(120–353) calculated from structure prediction with Phyre  between PDI(120–353) and BPA was calculated to be 1.17 × 10−5, 1.36 × 10−5, and 1.09 × 10−5m−1 at a scan rate of 60, 120, and 200 °C h−1, respectively (m) . These results suggest that only PDI(120–353) enhances the thermal stability due to BPA binding, but BPA does not have any effect on PDI(354–509).The dissociation constant ectively . The dise of BPA C. These A shows the molecular diameters of PDI(120–353) and PDI(354–509) measured at different molar ratios of BPA/PDI (ranging from 0 to 10). No significant change was observed in the mean diameter of PDI(354–509) in this range of BPA/PDI molar ratios ; however, the molecular size of PDI(120–353) markedly decreased upon titration of BPA up to a BPA/PDI molar ratio of ∼5 .To gain further structural insights into the BPA-bound form of PDI(120–353), we performed DLS and SAXS analyses. DLS is often employed to measure the size of target particles . Fig. 9AFig. 9q value of 0.10 Å−1 (B), suggesting a significant conformational change upon BPA binding. The apparent radius of gyration, gR(c), and the normalized forward intensity, I(0)/c, showed only marginal protein concentration dependence in the range of 0–5 mg/ml (C and D). The extrapolated gR value at c = 0 was 26.7 Å for the PDI(120–353)-BPA complex and 28.0 Å for PDI(120–353) alone. Thus, PDI(120–353) assumes a more compact structure when it binds BPA. This is consistent with the DLS observations, where the molecular diameter of PDI(120–353) markedly decreased upon the addition of BPA.To determine the structural parameters and the molecular shape of BPA-bound and -unbound forms of PDI(120–353), SAXS analysis was performed in the presence or absence of BPA. Notably, there was a striking difference between the scattering curves of the PDI(120–353)-BPA complex and PDI(120–353) alone around the 0.10 Å−1 B, sugges–5 mg/ml , C and DP(r) (E). Because P(r) indicates the distribution of linear distances between every pair of atoms in a particle, the most frequent r value and the largest r value (Dmax) can be determined from this function. The Dmax values were thus estimated to be 82.0 Å for PDI(120–353) alone and 79.5 Å for the PDI(120–353)-BPA complex, corroborating the present finding that BPA binding results in a more compact overall shape for PDI(120–353).To extract information on the geometrical shape of PDI(120–353), we calculated a pair distribution function, P(r) E. Becausin vitro. Consistent with the findings, BPA slowed down the reoxidation of PDI in HeLa cells, leading to the accumulation of a large amount of PDI in the reduced form. Thus, BPA can have an enormous influence on the properties of PDI both in vitro and in cells.The present study determined that BPA, an endocrine disruptor, markedly inhibited oxidative protein folding catalyzed by the Ero1α-PDI combination a domain, resulting in more compact overall shape. This conformational change probably led to inhibition of the capture of partially folded or misfolded proteins by PDI. Accordingly, the BPTI refolding assay clearly demonstrated that BPA inhibited the late stages of protein folding (and early stages to a lesser extent) from kinetically trapped intermediates to a native state . In this context, several lines of evidence suggest the significant plasticity of the bb′ segment of PDI. Crystal structures of reduced and oxidized forms of human PDI showed a redox-dependent domain arrangement of the b domain relative to b′  and PDI (A). Meanwhile, BPA significantly inhibited Ero1α-catalyzed oxidation of ERp46 although with a lower potency (IC50 = 538 μm) than that of PDI (IC50 = 41 μm) (C and 6C). The large difference in the effects of BPA on Ero1α- and Prx4-driven oxidative pathways presumably reflects the completely different mode of interaction of these two oxidative enzymes toward PDI family members. Systematic studies indicate that Prx4 oxidizes the thioredoxin domains of PDI family members at almost the same rate regardless of whether they are present on an isolated peptide or as part of an intact protein  C and 6C. protein , 28. Ero element , 23. WheIn vivo, BPA induces heat shock protein 90α expression in mouse uterus (An increasing number of studies have investigated the physiological effects of BPA on mammalian tissues. e uterus  and indue uterus . Also, Be uterus , which ie uterus . Of notee uterus , 56. One


Francisella genus comprises Gram-negative facultative intracellular bacteria that are among the most infectious human pathogens. A protein of 14.7 KDa named as FTN_1133 was previously described as a novel hydroperoxide resistance protein in F. tularensis subsp. novicida, implicated in organic peroxide detoxification and virulence. Here, we describe a structural and biochemical characterization of FTN_1133. Contrary to previous assumptions, multiple amino acid sequence alignment analyses revealed that FTN_1133 does not share significant similarity with proteins of the Ohr/OsmC family or any other Cys-based, thiol dependent peroxidase, including conserved motifs around reactive cysteine residues. Circular dichroism analyses were consistent with the in silico prediction of an all-α-helix secondary structure. The pKa of its single cysteine residue, determined by a monobromobimane alkylation method, was shown to be 8.0±0.1, value that is elevated when compared with other Cys-based peroxidases, such as peroxiredoxins and Ohr/OsmC proteins. Attempts to determine a thiol peroxidase activity for FTN_1133 failed, using both dithiols  and monothiols (glutathione or 2-mercaptoethanol) as reducing agents. Heterologous expression of FTN_1133 gene in ahpC and oxyR mutants of E. coli showed no complementation. Furthermore, analysis of FTN_1133 protein by non-reducing SDS-PAGE showed that an inter-molecular disulfide bond (not detected in Ohr proteins) can be generated under hydroperoxide treatment, but the observed rates were not comparable to those observed for other thiol-dependent peroxidases. All the biochemical and structural data taken together indicated that FTN_1133 displayed distinct characteristics from other thiol dependent peroxidases and, therefore, suggested that FTN_1133 is not directly involved in hydroperoxide detoxification. Francisella genus, a group of Gram-negative facultative intracellular bacteria, comprises species that are among the most infectious human pathogens. Indeed, F. tularensis subsp. tularensis can infect human airways as few as 10 c.f.u., and if untreated, generally provokes a fatal outcome F. tularensis subsp. novicida, holarctica and mediasiatica, in addition to other species, F. philomiragia, display reduced virulence to humans and rarely result in fatalities, acting like opportunistic pathogens F. tularensis can infect many host cell types that include epithelial, endothelial, polymorphonuclear neutrophils and mononuclear phagocytes F. tularensis is able to prevent the oxidative burst by inhibition of NADPH oxidase complex (NOX2) activity, the main Reactive Oxygen Species (ROS) generation machinery of the phagocytic cell F. tularensis seems to preferentially utilize mechanisms dedicated to inhibit ROS generation by the NADPH oxidase complex, some proteins directly involved in ROS decomposition are also recruited during the infectious process F. tularensis genome revealed the occurrence of genes that are directly involved in ROS detoxification. For example, in the genome of Francisella tularensis subsp. novicida U112, it is observed the presence of sodB and sodC genes ; katG, which encodes a bifunctional catalase/peroxidase enzyme; FTN_0698, which encodes a glutathione peroxidase (GPx); and FTN_1624, a member of DyP-type peroxidase family Bacterioferritin Comigratory Protein) protein that belongs to a BCP/PrxQ sub-family, according to a classification proposed by Francisella mutants of sodBC and katG genes have been shown to present reduced virulence Although F. tularensis subsp. novicidaBacillus megaterium.FTN_1133 was recently identified in a macrophage replication screen as a protein involved in virulence and also as a novel hydroperoxide resistance protein in Organic Hydroperoxide Resistance protein) belongs to a large family of proteins called Ohr/OsmC, which is subdivided into three sub-families: Ohr, OsmC and YhfA Ohr  medium at 37°C with appropriated antibiotics: ampicilin (100 µg/mL) or kanamycin (15 µg/mL).FTN_1133 gene was commercially synthesized by GenScript USA Inc. containing the sites for NheI and XhoI restriction enzymes in the flanking regions. To clone into pET15b expression vector (Novagen), FTN_1133 sequence was amplified by PCR, from pUC57 containing FTN_1133 sequence, using the following primers CATATGGCTATTAGTCAAAATGTTATAAAAATAC-3′5′-CGATC and GGATCCTTAGCTTTTATTATCGATCAAGCTTCG-3′5′-CGC, which contained sites for NdeI and BamHI restriction enzymes (bold letters). FTN_1133 gene was then sequenced using T7 promoter and terminator oligonucleotides. Cloning, expression, and purification of recombinant OsmC, from E. coli and Ohr, from Xyllela fastidiosa, were performed as described previously in Search for FTN_1133 homologous was performed using the PSI-BLAST 600 nm = 0.5) of E.coli AD494 (DE3) harboring the pET15b-FTN_1133 plasmid. Incubation was performed at 20°C with shaking and after 16 hours, cells were harvest by centrifugation and suspended in lysis buffer . Cells were disrupted by sonication  and debris was separated from supernatant by centrifugation at 15.000 rpm at 4°C for 40 minutes. The supernatant was filtered using a 0.45 µm pore membrane and applied using a peristaltic pump into HiTrap TALON crude column  containing agarose-Co++ resin. The column was washed sequentially with 3 column volumes of washing buffer  containing 50 mM and 100 mM of imidazole and eluted with 3 column volumes of elution buffer . Buffer exchange and concentration of purified proteins was done using Amicon Centrifugal 10 MW devices (Millipore). His-FTN_1133 purity was checked by SDS-PAGE analysis and the concentration was spectrophotometrically determined by its absorbance at 280 nm  in exponential culture (ODCircular Dichroism (CD) spectra were collected on a JASCO J-710 CD spectrometer using 3 or 6 µM of FTN_1133 in the presence of 100 mM NaCl, 20 mM sodium phosphate pH 7.4 and 10% glycerol in a 0.01 cm cell. Each CD spectrum resulted from the accumulation of 8 scans collected at scan rate of 20 nm/min with 1 nm band width and a time constant of 1 s. Data were collected from 260 to 195 nm and deconvoluted with CDpro-package software that uses SELCON3, CONTINLL, CDSSTR and CD CLUSTER algorithms 2O2was monitored by FOX assay Diethylene Triamine Pentaacetic Acid) (0.1 mM), sodium azide (0.1 mM), DTT (0.5 mM), β-mercaptoethanol (5 mM) or reduced glutathione (GSH) (5 mM) and CuOOH, tBOOH or H2O2 (200 µM) were mixed in 50 mM Hepes-HCl buffer, pH 7.4  at 37°C during 0, 10, 20 and 40 minutes. Reactions were started by addition of hydroperoxides and stopped by addition of 20 µL of 5 M of HCl. Afterwards, reactions mixtures were diluted into 0.9 mL of FOX solution  and incubated during 30 minutes at 37°C. The absorbance values were recorded at 560 nm and concentrations were calculated according to calibration curve previously performed for all tested peroxides.Reduction of cumene hydroperoxide (CuOOH), t-butyl hydroperoxide (tBOOH) or HE.coli (Sigma-Aldrich #T0910). Formation of oxidized DTT was followed by its intrinsic absorbance at 310 nm (ε310 nm = 110 M−1.cm−1) E.coli (1 µM), DTPA (1 mM) and CuOOH or tBOOH (2 mM) in 100 mM of sodium phosphate buffer, pH 7.4. Reactions were initiated by addition of DTT (10 mM).In the oxidized DTT assay, the peroxidase activity was assessed in the presence and absence of the Thioredoxin (TrxA) from Peroxidase activities were also analyzed by the lipoamide/Lipoamide dehydrogenase and GR/Grx/GSH system coupled assays. In these cases, rates were determined by decay of absorbance at 340 nm, as a consequence of NAD(P)H oxidation X. fastidiosa) (0.5 µM) and NADH (200 µM) in 50 mM of sodium phosphate buffer, pH 7.4, were initiated by addition of CuOOH, tBOOH or H2O2 (200 µM) For the lipoamide/Lipoamide dehydrogenase coupled assay, reaction mixtures of 1 mL  containing FTN_1133 (10 µM) or OsmC (10 µM) in the presence of reduced lipoamide (50 µM), DTPA (100 µM), Lpd (Dihydrolipoamide dehydrogenase from Hydroxy Ethyl Disulfide) (0.7 mM) and GSH, when incubated at 30°C for 3 min. Reactions were started by the addition of GrxC and followed by the decrease in the absorbance at 340 nm due to the oxidation of NADPH For the GR/Grx/GSH coupled assay, reaction mixtures of 1 mL  contained FTN_1133 (5 µM), yeast Glutathione Reductase (GR) (6 µg/ml), Glutaredoxin C (GrxC) (10 µM), GSH (1 mM), bovine serum albumin (BSA) (0.1 mg/ml), DTPA (2 mM) and NADPH (0.2 mM) in 100 mM Tris–HCl buffer, pH 7.4. Reactions were initiated by addition of CuOOH or tBOOH (0.2 mM). GR and GrxC activities were previously assayed by their capacity to reduce the mixed disulfide between HED  an ahpC E. coli BW25113 mutant, which was deficient in response to organic hydroperoxide and (ii) an oxyR E.coli BW25113 mutant, which was deficient in response to H2O2 and to a lower extent to organic hydroperoxide challenge, as assessed by the disk inhibition assay (unpublished data).Two mutant strains of FTN_1133 or osmC (positive control) genes were grown until OD600 nm = 0.5, induced by addition of 0.5 mM of IPTG for two hours and plated on LB supplemented with ampicillin (100 µM) and IPTG (100 µM). After that, 6 mm paper filter disks were saturated with 10 µL of solution of tBOOH (75 mM), CuOOH (100 mM) or H2O2 (500 mM) and placed on the surface of inoculated LB plates. After 16 hours, the size of inhibition zone was measured with a ruler. The statistical analyses were performed between wild type and mutant strains carrying the pPROEX-FTN_1133 plasmid and analyzed by the unpaired Student's t-test. In each graph, error bars represent the standard deviation (n = 9).To test if FTN_1133 was capable to complement the organic peroxide sensitive phenotype, mutant strains that harbored pPROEX expression vectors containing a of the cysteine residue from FTN_1133 was determined by a monobromobimane (mBrB) alkylation method that generates a fluorescent product, detected at λexc 396 nm and λem 482 nm Morpholino Ethanesulfonic Acid] and 30 mM of Tris-HCl buffer adjusted to the pH values from 3.5 until 9.0. The assays were performed in flat-bottom white polystyrene 96-well plates (Costar) as triplicates, using Eclipse Varian Spectrofluorimeter, operating at medium voltage with both emission and excitation slit of 5 nm. As control of method, the same procedure was performed with recombinant Ohr protein from Xylella fastidiosa, and OsmC protein from E. coli. An additional blank reaction was performed in absence of thiols, to determine whether buffer components could interfere with the reaction or not.The pKThe angular coefficients were calculated using time points that included at least the initial 10 min of reaction that were fitted in a straight line. The plots displayed were fitted by non-linear regression to Henderson-Hasselbalch equation considering 95% of confidence using Prism 4 for Windows, GraphPad Software, San Diego, CA.2O2 was analyzed by non-reducing SDS-PAGE. Aliquots of FTN_1133 (5 µM) were treated with 1, 5, 10, 20 50 or 100 µM CuOOH, tBOOH or H2O2 in a buffer containing NaCl (0.1 M) and DTPA (1 mM) in 20 mM of sodium phosphate buffer, pH 7.4, for ten minutes at 37°C. Immediately after that, samples were treated with 100 mM with NEM  for one hour at room temperature.Induction of inter-molecular disulfide bond in FTN_1133 by organic or by H2O2 (100 µM) in a sodium phosphate (20 mM) buffer, pH 7.4 containing NaCl (0.5 M) and DTPA (1 mM) for 0, 0.5, 1, 2, 3, 4, 18 and 22 hours at 30°C and after each time point, the remaining cysteine sulfhydryl groups were alkylated with NEM (100 mM) for one hour at room temperature. As control of experiment, aliquots of FTN_1133 (5 µM) were submitted to the same experimental conditions, with no addition of hydroperoxides.In another experiment, aliquots of FTN_1133 (5 µM) were treated with CuOOH, tBOOH or HAfter all treatments, the respective samples were separated in a 14% non-reducing SDS-PAGE gel and stained by Comassie Blue.FTN_1133 is a protein with 127 amino acid residues and, in contrast to the Ohr/OsmC proteins (which depends on two cysteine residues for its peroxidase activity), has only one cysteine residue . FTN_113Francisella tularensis were retrieved  and poor coverage (37%). The lack of significance between FTN_1133 and Ohr family was corroborated by performing a PSI-blast analysis using OhrA sequence from Bacillus megaterium as query. This inverted analysis did not retrieve FTN_1133 sequence, even after 10 iterations (data not shown). Additionally, FTN_1133 sequence was not present among 7485 sequences belonging to the Ohr/OsmC family in the pFAM database.It is worthwhile to mention that a PSI-blast search using FTN_1133 sequence as query, retrieved one sequence of OhrA from Therefore, it is not appropriate to consider FTN_1133 a member of the OsmC/Ohr family. We characterized the recombinant FTN_1133 structural and biochemically in attempt to understand its possible function as a thiol peroxidase.in silico’ prediction, we performed circular dichroism analysis of FTN_1133 recombinant protein. CD spectra of FTN_1133 display minima at 208 nm and 222 nm, characteristic of proteins that have only α-helix and not β-sheet  that remained in the reaction mixtures, after incubation of substrate with FTN_1133 in the presence of DTT. FTN_1133, even at high concentrations, was not able to degrade CuOOH or tBOOH faster than the uncatalyzed reaction (absence of protein), even after 40 minutes of reaction  genes were cloned into pPROEX expression vector. The expression of the recombinant proteins FTN_1133 and OsmC in E. coli ahpC and oxyR mutant strains were checked by western blot  or OsmC (2.06±0.27 cm)   resulteda that is related with the stabilization of the transition state a value of the single Cys residue of FTN_1133 by the monobromobimane alkylation method All the Cys-based thiol peroxidases so far described carry reactive cysteine, whose thiolate groups display an acidic pKa value ranged from 7.7 to 8.2, which can be considered high when compared to values observed for reactive Cys residues from proteins whose thiol dependent peroxidase activities are well established , we decided to study the thiol redox state of its cysteine residue in response to peroxides based on previously published methods 2O2 for ten minutes at 37°C and no inter-molecular disulfide bond formation was observed , when compared with the control reaction, where no hydroperoxide was added  of CuOOH, tBOOH or HFrancisella tularensis subsp. novicida mutant for FTN_1133 gene presented a reduced capacity to detoxify organic hydroperoxides in vivo−1 s−1, that is several orders of magnitude lower than similar rate constants (106–108 M−1 s−1) of other thiol peroxidases Francisella genus, we raised the hypothesis that FTN_1133 could indirectly participate in a particular peroxidase system restricted to bacteria belonging to the Francisella genus. In support to this notion, AhpD (a thioredoxin-like protein) provides electrons to AhpC, a Cys based peroxidase from Mycobacterium tuberculosisFTN_1133 gene, we observed the occurrence of two neighborhood genes FTN_1132 and FTN_1134, whose functions were not assigned yet. FTN_1132 was annotated as a hypothetical protein and its occurrence is restricted to Francisella genus (like FTN_1133 does). FTN_1134 presents a conserved Sdh5 domain that is involved in flavinylation and activation of Succinate Dehydrogenase (SDH) complex in both eukaryotes and bacteria Flavin Adenine Dinucleotide), a redox active molecule, it is tempting to speculate that FTN_1134 could be an electron donor required for a peroxidase system that FTN_1133 might participate.Taken together, the observed structural and biochemical features presented here for FTN_1133, contrast with those presented for other known thiol dependent peroxidases, including proteins belonging to the Ohr/OsmC family. Since orthologous proteins to FTN_1133 are restricted to Francisella virulence FTN_1133 was clearly shown to be implicated with Figure S1Assay for FTN_1133 thioredoxin-dependent peroxidase activity. Peroxidase activity was followed by generation of oxidized DTT. A. and B. DTT oxidation by CuOOH or tBOOH, respectively. Reactions were carried out in the presence of FTN_1133 (5 µM), sodium phosphate pH 7.4 (100 mM), DTPA (1 mM) and organic hydroperoxide (2 mM), with (red line) or without (green line) addition of 1 µM of recombinant TrxA from E. coli, and started by addition of 10 mM of reduced DTT. As positive control of reaction, the same assay was made using 10 µM of OsmC without addition of TrxA (blue line). The blank reaction (black line) was performed without any enzyme addition. The figure is representative of at least two independent set of experiments.(TIF)Click here for additional data file.Figure S2Assay for FTN_1133 lipoyl-dependent peroxidase activity. Lipoamide-Lipoamide dehydrogenase coupled assay was followed decay of absorbance at 340 nm due to NADH oxidation. A., B. and C., represent NADH oxidation in the presence of CuOOH, tBOOH and H2O2 at 37°C, respectively. Reactions were performed with FTN_1133 (10 µM), sodium phosphate pH 7.4 (50 mM), of reduced lipoamide (0.05 mM), DTPA (1 mM), recombinant Lpd (0.005 mM) from Xyllela fastidiosa under gently agitation (red line). After one minute for temperature stabilization, 0.2 mM of NADH was added to reaction that was finally started by addition of 0.2 mM of respective hydroperoxide. As positive control of reaction (blue line), the same assay was carried out using 10 µM of OsmC. The blank reaction (black line) was performed without any enzyme addition. The figure is representative of at least two independent set of experiments.(TIF)Click here for additional data file.Figure S3Assay for FTN_1133 Grx/GSH-dependent peroxidase activity. GR/GSH coupled assay was followed by NADPH oxidation. A. and B., NADPH oxidation in the presence of CuOOH and tBOOH at 37°C, respectively. The reaction containing Tris–HCl pH 7.4 (100 mM), yeast GR (6 µg/ml), GrxC (10 µM) from X. fastidiosa, GSH (1 mM), BSA (0.1 mg/ml), DTPA (2 mM) and NADPH (0.2 mM) was initiated by addition of 0.2 mM of CuOOH or tBOOH. C. For GR and GrxC activity control reaction, FTN_1133 and hydroperoxide, were substituted by 0.7 mM of HED that was incubated at 30°C for 3 min for the formation of the mixed disulfide between GSH and HED. The reaction was started by the addition of GrxCx.f. and followed by the decrease in the absorbance at 340 nm due to the oxidation of NADPH (TIF)Click here for additional data file.Figure S4Expression analysis of recombinant FTN_1133 and OsmC proteins in wild type (BW25113), ΔahpC and ΔoxyR backgrounds.A. and C. Comassie stained gels of total extracts from IPTG induced cultures of wild type BW25113 (lane 2), ΔahpC (lane 4) and ΔoxyR (lane 6) strains, which harbored pPROEX-FTN-1133 or pPROEX-OsmC constructions, respectively. As control, the same strains harboring the empty vector were also induced . B. and D. Western blot analysis of the same extracts used in A. and C. The order of WB lanes was the same presented for Comassie stained gels. Histidine Tag (6×His) Monoclonal Antibody (Novex) was used to detect His-tagged proteins.(TIF)Click here for additional data file.