Aspergillus fumigatus, an opportunistic human fungal pathogen. There are three genes  controlling the biosynthesis of α--glucan in this fungal species. Deletion of all the three AGS genes resulted in a triple mutant that was devoid of α--glucan in its cell wall; however, its growth and germination was identical to that of the parental strain in vitro. In the experimental murine aspergillosis model, this mutant was less pathogenic than the parental strain. The AGS deletion resulted in an extensive structural modification of the conidial cell wall, especially conidial surface where the rodlet layer was covered by an amorphous glycoprotein matrix. This surface modification was responsible for viability reduction of conidia in vivo, which explains decrease in the virulence of triple agsΔ mutant.α--Glucan is a major component of the cell wall of  Aspergillus fumigatus is the predominant mold pathogen of humans, responsible for life-threatening systemic infections in patients with depressed immunity. Because of its external localization and specific composition, the fungal cell wall represents a target for recognition by and interaction with the host immune cells. In A. fumigatus, α--glucan is a key component of the extracellular matrix, which encloses the cell wall β--glucan-chitin fibrillar core. Interestingly, the deletion of the genes responsible for α--glucan synthesis resulted in a mutant that exhibited wild type phenotype in vitro; while the altered cell wall organization resulted in this fungus being avirulent in vivo. This study confirms that any modification in the cell wall components is associated with compensatory reactions developed by the fungus to counteract stress on the cell wall that may result in unexpected fungal response when challenged with the host immune system. Paracoccidioides brasilisensis, Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans Aspergillus fumigatus). The role of this polysaccharide during infection has been demonstrated and the mechanisms of its involvement in establishing virulence have been forwarded C. neoformans, α--glucan anchors the capsule, a well known virulence factor of this fungus, to the yeast cell wall and has been shown to be indirectly associated with virulence since a mutant devoid of α- -glucan did not have any capsule and, most importantly, was unable to grow at 37°C H. capsulatum, α--glucan was suggested to be essential for virulence because it masked immunogenic molecules: in the α--glucan synthase mutant, β--glucan that is recognized by Dectin-1, is exposed at the surface of the cell wall, whereas in the parental strain yeast cells, β--glucan is covered by α--glucan, preventing Dectin1-dependent immune response α--Glucan is a major cell wall component of most ascomycetous and basidiomycetous fungi, including the human pathogens that establish their disease upon inhalation of their infective morphotypes -glucan accounts for 40% and 19% of the mycelial and conidial cell wall polysaccharides, respectively in vivo data were confirmed by in vitro experiments where dendritic cells pulsed with α--glucan induced Il12p70 production, a classical Th1 promoting cytokine A. fumigatus, this polysaccharide is synthesized by three α--glucan synthases (Agsp) AGS1, AGS2 and AGS3 genes was recently generated in our lab that resulted in an A. fumigatus mutant lacking α--glucan in the cell wall. In contrast to other fungal pathogens, this triple AGS A. fumigatus deletion mutant did not show a distinct growth phenotype in vitroIn ags1Δags2Δags3Δ (agsΔ) mutants devoid of α--glucan were used to investigate the role of α--glucan in A. fumigatus infection. As shown here, the virulence of these A. fumigatus triple agsΔ mutants was extremely attenuated in both immunocompetent and immunocompromised murine models of experimental aspergillosis tested. The defect in virulence correlated with a lack of vegetative fungal dissemination in the lungs, associated with a highly reduced inflammation following conidial inoculation. Analysis of the conidia of the triple mutants showed that the lack of virulence of the mutants in vivo was associated to major changes occurring on the cell wall, especially on the surface of the resting and swollen conidia, which resulted in an increased killing by phagocytes.In the present study, three independently constructed triple agsΔ mutants per lung was much lower than the CFUs per lung of the parental ku80 strain , cationic peptides, hypoxia and depletion of iron. The agsΔ mutants showed similar growth rates as their parental strain in the presence of Menadione, hydrogen peroxide and Luperox®101 with minimum inhibitory concentrations (MIC) of 30 µM, 10 mM and 2 µM, respectively (data not shown) irrespective of the pH of the medium (pH 7 or 4). The killing of resting conidia after 2–6 h of incubation with macrophages purified from uninfected p47phox−/− mice (depleted in ROS production) were similar to the killing by purified macrophages from uninfected wild type mice (C57BL6 H-2b) . In vitro, the agsΔ conidia germinated like parental strain conidia in culture medium without supplementation with iron as well as under hypoxic conditions (<1% (v/v) O2 and 9–13% (v/v) CO2). The agsΔ mutants were not more susceptible than the parental strain to cationic peptides. At doses of 230, 100, 40 and 230 µg/ml of Cathelicidin LL-37, α HNP2 and β hBD2 defensins and lactoferrin, respectively, no germination differences were seen between parental and mutant strains (data not shown). Similarly, both mutant and parental strain conidial killing was comparable with 0.05% SDS (data not shown). In addition, no increase in the intracellular labeling of the agsΔ mutant conidia was seen after incubation with Calcofluor White or FITC (data not shown). These results suggested that the agsΔ conidia were not more permeable to extracellular toxic molecules than the parental strain. Testing of these different inhibitors in combination  did not result in a differential sensitivity between the parental and mutant strains (data not shown).To investigate the mechanisms responsible for the L6 H-2b) . These rin vitro, the triple agsΔ mutants were not more susceptible to environmental stresses and antifungal molecules compared to the parental strain. To further investigate the differences in virulence between the mutant and parental strains in vivo, we hypothesize that the killing of the agsΔ mutant conidia could be due to the induction of an early and strong host immune response towards the mutant conidial morphotypes.These results suggested that, agsΔ mutant were more efficiently phagocytosed by mouse alveolar macrophages than that of the parental ku80 strain. After 1 h incubation, an average of 3.4 and 1.4 conidia of agsΔ mutants and ku80 were engulfed per macrophage, respectively . In contrast to the ku80 conidia that are covered with a crystalline-like array of rodlets agsΔ mutant conidial surface was amorphous without any organized structure  to extract the rodlet protein. Similar amount of the hydrophobic RodA protein, which constitutes the rodlet layer, could be extracted from the agsΔ and parental strain conidia . agsΔ and ku80 resting conidial HF-extracts. These data confirmed AFM and TEM observations that on the agsΔ mutant conidial surface the rodlets were present but hidden by an amorphous material.To investigate if the rodlet layer is still present on the ku80 and agsΔ mutant conidia by AFM using bare Si3N4 tip. agsΔ mutant conidial surface was associated with a dramatic reduction in their conidial surface adhesive properties. For the parental strain, force-distance curves recorded across the cell surface revealed large adhesion forces, with a magnitude of 0.6±0.039 nN as shown by the adhesion force histogram -glucan with the β--glucan receptor GNBP3, chitin with WGA, galactomannan (GM) with an anti-GM monoclonal antibody and galactosaminogalactan (GAG) with an anti-GAG monoclonal antibody were negative (data not shown). In contrast, a strong labeling of the resting agsΔ conidium with ConA was observed suggesting that the surface layer was rich in glyco-conjugates . Further, extracted protein mixture was subjected to proteomic analysis. Thirty-four proteins were identified and in-silico analysis of these proteins by SigPred  and CADRE (http://www.cadre-genomes.org.uk/Aspergillus_fumigatus/) revealed that all of them had a signal peptide except Sod1  and Aspf13 (AFUA_2G12630) agsΔ conidial surface material, indicating that the proteins recovered were not depending on the extraction buffer (data not shown). The fact that many proteins were present above the surface rodlet layer suggested that in contrast to the parental strain, the lack of α1,3 glucan has led to a different cell wall retainment of these glycoproteins in the agsΔ mutant conidia.To extract these amorphous surface materials, In vitro analysis of the cytokines produced during the first 5 h of incubation with alveolar macrophages showed that high amounts of pro-inflammatory TNFα cytokine were produced upon interaction with agsΔ mutant conidia whereas no TNFα was produced when the parental strain was incubated with macrophages under the same incubation conditions -glucan receptor GNBP3, whereas both resting and swollen conidia of the parent strain were negative -glucan, respectively, than the cell wall of the parental strain Increased cytokine production seen in the macrophages over a 5 h-time period could also come from changes occurring at the surface of germinating conidia since it has been shown previously that conidia starts germinating intracellularly in the macrophage lysosome after the first 2 h of phagocytosis tibodies . These ragsΔ mutant and parental strain conidia and their differential impact/in vivo fate based on our in vitro assays as well as in vivo experiments using murine aspergillosis models. The presence of glycoproteins hiding the rodlet layer increases the phagocytic rate and promotes an immediate host immunological response towards the triple agsΔ mutants during phagocytosis. Once the mutant conidium is internalized, the conidial swelling results in an increased exposure of PAMPs on the swollen agsΔ conidial surface. Such surface modifications further boosts pre-existing host defense induced by the resting agsΔ conidia. In contrast, the resting conidium of the parental strain are not recognized by the phagocytes and do not display major PAMPs on the surface of the conidium during the intracellular swelling. Since agsΔ conidia did not seem more sensitive to host antifungal molecules compared to the parental strain, we hypothesize that differences in the killing in the later growth stages resulted from an early and enhanced host response induced by the modified surface of the resting agsΔ conidia. This early stimulation will be responsible for the killing of the germinating agsΔ conidia. On the contrary, in the partially immunosuppressed experimental murine models, limited and delayed killing of the parental strain conidia enables their further vegetative growth.agsΔ mutants displayed a reduced virulence associated with an inhibition of germination in vivo and a reduction of the inflammatory response after 24 h infection (decreased TNFα and increased IL10 expressions and reduced recruitment of PMNs). The low level of TNFα seen with the triple agsΔ mutants fits with the lack of recruitment of neutrophils seen with this mutant after 24 h infection. However, during our in vitro experiments with macrophages incubated during 5 h with agsΔ or ku80 conidia, we observed the induction of pro-inflammatory cytokines. This indicated that the lack of inflammation seen at later stages of infection in mice was due to the inhibition of vegetative growth of the agsΔ mutants rather than a failure to stimulate inflammation. This was in agreement with the fact that agsΔ conidia were killed before their hyphal development.In this study we showed that the agsΔ mutants was the absence of visible rodlet layer on the conidial surface. Even though the rodlets were present in the mutant conidia, their masking by a (glyco-)protein layer restored the immune sensing that is usually silenced when the rodlets are present on the surface of the wild type conidia agsΔ conidia were covered by proteins, which are usually secreted during vegetative growth. Most hydrolases found in the additional amorphous surface layer of the resting agsΔ conidia were usually identified during mycelial growth in a protein-based medium AGS deletions. Interestingly, in three independent HF extractions, the amount of 14.5 kDa RodAp was slightly higher than the 16 kDa RodAp . Alternatively, the hydrolases, because of their enzymatic activity, may harm the cell wall structure itself and this would help the proteins to cross the cell wall barrier. The stimulation of the expression of TNFα after incubation with macrophages (isolated from naive mice BAL) with agsΔ mutant conidial NaCl extract showed that these proteins located on the conidial surface were sensed first by the immune system and were able to induce an immediate immune response towards agsΔ conidia. It was previously shown that some of these surface proteins are recognized by T cells and can induce a Th1 protective response agsΔ conidia conferred protection against infection, associated with a reduced neutrophil recruitment in BAL and a reduced inflammatory pathology in the lung. Hiding of the rodlet layer by an amorphous glycoprotein layer that stimulates the host response is not exclusively specific to the agsΔ deletion, since a similar conidial phenotype was observed on chitin synthase mutants B. dermatitidis, the absence of α--glucan at the surface of the yeast increased the expression of W1-1 adhesin/antigen that were bound to phagocytic cells and suppressed the generation of the pro-inflammatory cytokine TNFα The primary phenotype of the resting conidia of the AGS deletions also plays a role in stimulating the host innate immune response and inducing the production of antifungal molecules by the innate immune cells. The exposure of β--glucan at the surface of germinating agsΔ conidia will favor a Dectin-1-mediated host response A. fumigatusagsΔ mutants, it was shown that the lack of α--glucan in H. capsulatum also led to the unmasking of PAMPs B. dermatitidis and P. brasiliensis where the absence of α--glucan at the surface of the yeast and/or its replacement by β--glucan stimulated the host defense reaction Magnaporthe griseaThe exposure of polysaccharide PAMPs on the surface of germinating conidia consecutively to triple agsΔ conidia remain unknown. However, it is clear that ROS were not responsible for the differences in killing between the agsΔ mutants and the parental strain conidia since the agsΔ mutants did not display a higher sensitivity to ROS in vitro and the killing of agsΔ conidia was similar in p47phox−/− mice compared to C57BL/6 -glucan remains a two-sided coin. In the wild type strain, α--glucan induces an anti-A. fumigatus response as the injection of this polysaccharide into mice was immunoprotective and obviously responsible for the production of a Th1 response that is directed against A. fumigatusAGS deletions. The presence of glycoproteins hiding the rodlet layer and the exposure of PAMPs in the germinating conidia modified the immunological response of the host, which increased phagocytosis and killing of the agsΔ mutants, and induced pro-inflammatory cytokine production. It is the structural modification of the entire cell wall consecutive to the AGS deletions that is responsible for an early stimulation of the host defense reactions. Interestingly, these structural modifications did not modify the survival of the fungus in vitro but are essential for the in vivo survival. The difference in the surface composition of the resting and swollen conidia of the agsΔ mutants led to an immediate sensing of the immunogenic molecules resulting in an early response of the phagocyte towards the agsΔ conidia. The deleterious effect of a delayed immune response on the microbial virulence is well known.The story of AGS genes in this study but also with other cell wall genes or consecutively to the use of antifungals acting on the cell wall in several fungal species The α--glucan study tells us that the deletion of one cell wall gene does not lead only to the disappearance of the product of the encoded gene but results in a complete restructuration of the fungal cell wall. This has been shown with the deletion of the All strains were grown in 2% (w/v) malt agar slants and 1 week-old conidia were recovered from the slants by vortexing with 0.05% (v/v) Tween 20 aqueous solution. Swollen conidia and germ tubes were produced after 5 h and 10 h, respectively, after incubation at 37°C in Brian's medium (Brian) A. fumigatus parental strain AkuBku80ΔpyrG  obtained previously ags1Δags2Δags3Δn8and ags1Δags2Δags3Δ_n6.2 (agsΔ_n8 and agsΔ_n6.2), were used in this study. Since it had been impossible to complement agsΔ mutant for reasons explained previously agsΔ mutants were constructed independently using the strategy described previously to exclude the possibility that undesired mutations had occurred during the deletion process. The lack of α--glucan in the cell wall of mutant strains was confirmed by both chemical and immunolabeling assays . The sample immobilization is achieved by assembling the living conidia within the patterns of microstructured, functionalized poly-dimethylsiloxane  stamps using convective/capillary deposition 9 dry conidia with 48% (v/v) hydrofluoric acid (HF) for 72 h at 4°C. The contents were centrifuged  and the supernatant obtained was dried under N2. The dried material was reconstituted in H2O and an aliquot was subjected to 15% (w/v) SDS-PAGE analysis and visualized by silver nitrate staining. Bands were quantified using Image lab software (BioRad).The rodlet layer was extracted from the spore surface by incubating 1010 conidia per ml. The NaCl supernatant was recovered after centrifugation and directly subjected to 10% SDS-PAGE (w/v). The protein concentrations in the extracts were determined by the Coomassie brilliant blue method To analyze the components present on the surface, conidia were incubated in 0.5 M NaCl solution for 2 h at room temperature at a ratio of 104Cl in PBS, once with PBS and then incubated with different antibodies or lectins as described previously Resting and swollen conidia were PFA-fixed (2.5% (v/v) PFA in PBS) for one night at 4°C, washed three times with 0.1 M NHGalactosaminogalactan (GAG) was labeled with a monoclonal mouse antibody as described previously f) monoclonal antibody  at a concentration of 3 µg/ml and a polyclonal mouse antiserum against GNBP3 at 1∶200 dilution -glucan was labeled with the N-terminal β--glucan binding domain of The glucosamine moiety of chitin/chitosan and mannose/glucose moieties of glycoproteins and glucans were labeled respectively with WGA-FITC and ConA-FITC (Sigma) at 0.1 mg/ml concentrations upon incubating the conidia for 15 min at lab temperature.tert-butylperoxy)-2,5-dimethylhexane (Luperox®101) (0 to 2 mM) were tested on both parental and mutant A. fumigatus strains grown on agar-RPMI  supplemented with 1% agar (Difco), 0.3 g/1 L-glutamine and 0.1 M MOPS or MES  at 37°C for 24–48 h.Stress conditions induced by Menadione (0 to 30 µM) and 2,5-Bis were tested on A. fumigatus strains grown on Brian medium without supplementation with iron or RPMI-glutamine-MOPS medium (described above) 2O2 and Lactoferrin or Cathelicidin LL37 at concentrations of 231 µg/ml were tested in the same media, as described in Clavaud et alStress conditions induced by Lactoferrin 0.45–231 µg/ml (Sigma) or Cathelicidin LL-37 0.45–231 µg/ml (Sigma), SDS  and H6 conidia/ml with the defensins for 10–16 h at 37°C in RPMI-glutamine-MOPS medium.Stress condition induced by HNP2  and hBD2  defensins were also tested by incubating 10A. fumigatus strains was tested in Brian medium without supplementation with iron at 37°C and under hypoxia conditions using AnaeroGen sachet (Oxoid), which reduces the oxygen level in a jar to below 1% that results to a CO2 level between 9–13%.The growth of 4 in acetone for 1 h at −85°C before being slowly warmed to room temperature over 5 h. The cells were then rinsed in acetone and slowly infiltrated with and polymerized in Spurr's resin. Embedded cells were cut into serial 70 nm thick sections with an Ultracut R Microtome  and collected on Formvar-coated copper slot grids. Sections were post-stained with 2% uranyl acetate in 50% ethanol for 5 min followed by 5 min with Sato's lead citrate Aliquots (20 µl) of concentrated conidia were placed onto a Formvar-coated nickel or gold mesh grids, which were then placed between the flat sides of two B-type brass planchets . The grids were used as spacer creating a thin layer of cells that allows higher yields of well-frozen cells. The samples were immediately frozen with liquid nitrogen under high pressure  using a Bal-Tec HPM 010 high pressure freezing machine . Following cryofixation, the samples were freeze-substituted at −85°C in 1% glutaraldehyde  and 1% tannic acid in acetone for 72 h. After, the samples were rinsed thoroughly with three changes of fresh acetone at −85°C for a total of 45 min. Cells were infiltrated with 1% OsOb) mice were obtained from Charles River Breeding Laboratories . Experiments were performed according to the Italian Approved Animal Welfare Assurance A-3143-01. Breeding pairs of homozygous p47phox−/−mice, raised on C57BL6 background, were purchased from Harlan Laboratories and bred under specific-pathogen free conditions at the breeding facilities of the University of Perugia, Perugia, Italy E. coli β-galactosidase (clone GLL 113) was used as a control IgG. Treatment with the anti-Ly6G MAb is known to selectively deplete mature neutrophils, eosinophils and dendritic cells 3 compared to 1120±227/mm3 in controls, and the treated mice continued to be low for circulating neutrophils counts up to 5-days. Mice were monitored for survival and fungal growth (determined as colony forming unit (CFU) per organ) four days post-infection as described previously Female 8- to 10-week-old inbred C57BL6 (H-2b) and p47phox−/− mice were incubated at 37°C with unopsonized resting or swollen conidia (6½ h in RPMI at 37°C), at an effector to fungal cell ratio of 1∶10, for 2–6 hours in an ELISA plate wells. After removing the supernatant, Triton X100 (1%) was added to the wells and incubated at 37°C for 10 min to lyse the macrophages and to collect phagocytized conidia. The percentage of phagocytized conidia capable of further germination was determined by spotting phagocytized conidia (at suitable dilution) on a nutritive agar medium and counting those conidia capable of forming germ tube among spotted conidial population. We verified that the use of Triton X100 to lyse macrophage did not affect conidial germination as the percentage of germinations were similar (97±1%) for the agsΔ_5T, agsΔ_n6.2, agsΔ_n8 mutants and the parental strain with or without Triton-treatment. The differences in the germination of the conidia from the stock solution used for macrophage conidicidal activity study permitted the calculation of conidiocidal activity.For phagocytosis and conidiocidal activity, alveolar macrophages from uninfected mice were isolated from BAL as described agsΔ NaCl extracts containing 3.2 µg proteins, for 5 h. The cytokines expressed and productions were quantified by Real-time PCR and ELISA, respectively as described previously For cytokine quantification, total RNA was extracted from lungs of immunocompetent mice four days post-infection, or from macrophages isolated from BAL fluid of uninfected mice and incubating with p-values≤0.05 were considered to be significant. Data were representative of at least two independent experiments or pooled from three to five experiments. The in vivo groups consisted of six mice/group and experiments were repeated at least three times. Macrophage experiments were done three times with three different batches of macrophages and conidia.Statistical significance was analyzed by one- or two-way ANOVA or paired t-test with Prism software  and agsΔ_5T. Infections were performed under avertin anesthesia and all efforts were made to minimize suffering.Figure S1Immunocompetent mice infected with resting conidia of agsΔ triple mutants and parental ku80 strain. Observations and analysis on mice were done four days post-infection. (A) Fungal CFUs in lungs infected with conidia of agsΔ_5T, agsΔ_n6.2, agsΔ_n8 and ku80. (B) lung histology (periodic acid-Schiff-staining) and (C) Percentages of monocytes and polymorphonuclear cells found in the lung alveolar lavage (BAL) of mice infected with conidia of agsΔ_n6.2 and agsΔ_n8 mutants (periodic acid-Schiff-staining and Gomori's methanamine silver-staining) (D) Relative expression of TNFα and IL10 assessed by real time RT-PCR of the total RNA extracted from the lungs of naïve and mice infected with conidia of agsΔ_n6.2 and agsΔ_n8 mutants and ku80. Data are representative of at least three independent experiments. Ctl, naïve mice; *, P<0.05.(TIF)Click here for additional data file.Figure S2Survival of Cyclophosphamide immunosuppressed mice infected with resting conidia of agsΔ_n8 mutant and parental ku80 strains. The survival is expressed in percentage. Data are representative of at least three independent experiments.(TIF)Click here for additional data file.Figure S3Phagocytosis after 1 h incubation of agsΔ_n8 and parental ku80 resting conidia by the macrophages isolated from uninfected mice. Results expressed in number of conidia per macrophages. Data are representative of at least three independent experiments. *, P<0.05.(TIF)Click here for additional data file.Figure S4Imaging and adhesive properties of resting conidia of agsΔ_n8 mutant. Structural changes correlate with a loss of cell surface adhesive properties. (A) Height images ; (B) adhesion force maps (z-range: 5 nN) corresponding to the height image; (C) Representative force-distance curves and adhesion force histograms (n = 1024) recorded on the surface of agsΔ_n8 mutant conidia.(TIF)Click here for additional data file.Figure S5NaCl extracted proteins from the surface of the resting agsΔ triple mutant conidia. SDS-PAGE (10% gel) of proteins extracted after 2 h incubation of the resting conidia in 0.5M NaCl showing that the three triple agsΔ mutants  displayed the similar protein patterns.(TIF)Click here for additional data file.Figure S6TNFα production or expression by macrophages (isolated from uninfected immunocompetent mice) upon interaction with the parental strain ku80, agsΔ_n6.2 and agsΔ_n8 resting conidia, or the agsΔ_n8 and agsΔ_n6.2 conidial NaCl extract (3.2 µg proteins) respectively. (A) TNFα was quantified after 5 h macrophage-conidial interaction. (B) Relative expression of TNFα assessed by real time RT-PCR in total RNA from macrophages after 5 h incubation of the agsΔ_n8 and agsΔ_n6.2 conidial NaCl extract with macrophages. NaCl supernatant from ku80 resting conidia incubated for 2 h in 0.5M NaCl was used as a control. NS: Non-stimulated. *, P<0.05.(TIF)Click here for additional data file.Figure S7Immunolabeling of Galactosaminogalactan (GAG) and galactomannan (GM) on the swollen conidial surface of the triple agsΔ mutants and parental ku80 strains. Note that there is no differences in the amount of GAG (A)  and GM (B)  in the triple agsΔ mutant and parental strains.(TIF)Click here for additional data file.Figure S8Immunolabeling of α--glucan. Germinating conidia were labeled with MOPC that recognises α--glucan, and mouse TRITC conjugated anti-IgG was used as the secondary antibody. Note the absence of labeling on the triple agsΔ mutants - agsΔ_5T, agsΔ_n6.2 and agsΔ_n8.(TIF)Click here for additional data file.Table S1Identification of NaCl extracted conidial surface proteins from the agsΔ_5T and agsΔ_n8 mutants by MALDI-TOF/TOF.(1)Number of peptide peaks identified per protein.(DOCX)Click here for additional data file.


The ratio of the lengths of the second and fourth finger (2D∶4D) has beendescribed as reflecting the degree of prenatal androgen exposure in humans.2D∶4D is smaller for males than females and is associated with traits suchas left-handedness, physical aggression, attention-deficit-hyperactivitydisorder and a genetic polymorphism of the androgen receptor. All of thesetraits are known to be correlated to the vulnerability for alcohol dependency.We therefore hypothesized low 2D∶4D in patients with alcohol dependency.In the present study on 131 patients suffering from alcohol dependency and 185healthy volunteers, we found that alcohol dependent patients had smaller2D∶4D ratios compared to controls with preserved sexual dimorphism butwith reduced right-left differences. The detection of alcohol dependency basedon 2D∶4D ratios was most accurate using the right hand of males. These findingsprovide novel insights into the role of prenatal androgen exposure in thedevelopment of alcohol dependency and for the use of 2D∶4D as a possibletrait marker in identifying patients with alcohol dependency. Men have lower 2D∶4Dratios and consume more alcohol This study is part of the FLIP-project (Finger-Length In Psychiatry). BetweenFebruary 2009 and March 2010 we recruited 131 patients  who were treated as inpatients in theDepartment of Psychiatry in the Frankenalbklinik Engelthal. All patients sufferedfrom alcohol dependency according to ICD-10-criteria. Controls consisted of 185volunteers recruited predominantly from among employees of the University of Erlangen-Nurembergvia direct personal contact. Written informed consent was obtained after completedescription of the study to the subjects. The study was approved by the local EthicsCommittee (Ethik-Kommission der Medizinischen Fakultät der Friedrich-AlexanderUniversität Erlangen-Nürnberg).www.gimp.org) to measure the index(2D) and ring (4D) fingers from the hand scans. This technique provides goodreliability Scanning was conducted prior to examining or analyzing questionnaires scores. A HPscan-jet G4050 was used to scan participants' hands. To increase accuracy,small marks were drawn on the basal creases of the index and ring fingers beforescanning. Both hands were scanned at the same time, palms down. We used the GNUImage Manipulation Program  Reliability of the three raters was high for both the right hand  and the left hand . Reliability of the Dr-l values were also high(ICC = 0.898). The mean 2D∶4D and Dr-l values arepresented in 2 = 0.049), sex, hand*diagnosis-interaction and age. There were no further significantmain or interaction effects. The interaction line plots for 2D∶4D ratios formale and female subjects are shown in 2 = 0.014) with no further main or interactioneffects. Together, these results indicate that alcohol dependent patients havesmaller 2D∶4D ratios with preserved sexual dimorphism but with reducedright-left differences. The ROC analysis indicates highest diagnostic accuracy ofthe 2D∶4D ratio of the right hand in males , followed by males' left hand , females' right hand  and females' left hand .2D∶4D ratio was most associated, in descending order, with diagnosis As2D∶4D is fixed before birth We used a case control design without a matched sample approach. (1) There weredifferences in age between the patient and control groups. In agreement with Manninget al. 2010 May prolonged alcohol consumption have induced changes in 2D∶4D in later life?This appears unlikely, because (1) the differences in 2D∶4D between controlsubjects and alcohol dependent patients observed in our study are large. (2)Furthermore, it would be hard to explain, why alcohol should have different effectson the index finger compared to the ring finger. (3) In addition, duration ofalcohol use disorder (range: 0.5–44 years) was not related to 2D∶4D inthe patients investigated here when controlling for age (data not shown). However,in order to definitely answer this question would require a longitudinal study withindividual tracking of changes of 2D∶4D due to heavy alcohol consumption. Suchdata are currently not available.Alcohol consumption of the mother may have an impact on finger length ratios of theoffspring, as has been shown in rodents. Maternal alcohol consumption results inreduced testosterone levels and more female finger length ratios In conclusion, we were able to demonstrate that alcohol dependent patients have small2D∶4D ratios. Prenatal androgen exposure might be a missing link betweenseveral well-known findings in alcohol research. Further studies are needed toclarify the value of 2D∶4D as a diagnostic or prognostic marker for alcoholdependency.


We present this method as a fast and robust screening method for milk adulteration analysis and prevention of food intoxication.Nitrogen compounds like urea and melamine are known to be commonly used for milk adulteration resulting in undesired intoxication; a well-known example is the Chinese episode occurred in 2008. The development of a rapid, reliable and economic test is of relevance in order to improve adulterated milk identification. Cyclic voltammetry studies using an Au working electrode were performed on adulterated and non-adulterated milk samples from different independent manufacturers. Voltammetric data and their first derivative were subjected to functional principal component analysis (f-PCA) and correctly classified by the KNN classifier. The adulterated and non-adulterated milk samples showed significant differences. Best results of prediction were obtained with first derivative data. Detection limits in milk samples adulterated with 1% of its total nitrogen derived from melamine or urea were as low as 85.0 mg·L Louis, MO, USA) stock solution (100 mM) was prepared in buffer phosphate . For further experiments, the optimal conditions were set to a scan rate of 25 mV·s−1 and a potential window between −800 and +1,000 mV to linearly scan the Au WE versus the reference electrode. Cyclic voltammetries started and ended at −800 mV.The WE characterization was performed with a phosphate buffer solution between −800 and +1,300 mV and with a potassium ferricyanide solution between −800 and +1,500 mV. These experiments were done at room temperature following the general procedure described before (Section 2.2). A phosphate buffer solution  was prepared with 18.8 g·L2.4.M) were made with rehydrated powdered skimmed milk of two different brands: La Serenisima®  and Sancor®  with distilled water , as indicated by manufacturer's instructions (100 g·L−1). Replicated samples were prepared independently for quintuplicate (La Serenisima) or quadruplicate (Sancor).Milk samples , 3 g·L−1 of NaCl , and a nitrogen compound either urea  or melamine . The components of the “synthetic milk” listed above mimics some native components found in bovine milk, so their concentration resembles the concentration of their respective components. These samples have the same nitrogen molar concentration than bovine milk. “Synthetic milk” samples were named as urea “synthetic milk” (0USM) or melamine “synthetic milk” (0MSM) depending on which nitrogen compound was used to prepare them. All samples were prepared daily. Melamine is slightly soluble in water so 0MSM was prepared with hot distilled (milliQ) water (80 °C) and maintained at 40 °C to increase melamine solubility.“Synthetic milk” samples were based on an aqueous solution of distilled water  containing 49 g·L0USM or MSM0 to M to simulate series of gradual adulterated samples  of 85 g·L−1 . Conduct2.5.0USM and MSM_low level contains adulterated milk with 1, 2 and 5% of 0MSM. The USM_high level contains adulterated milk with 10 and 20% of 0USM and MSM_high level contains adulterated milk with 10 and 20% of 0MSM. A “leave-one-out” strategy was applied to validate the KNN classifier.The sensor outputs (cyclic voltammograms) collected by the potentiostat were imported to a personal computer and the statistical analysis was performed with R software (R Development Core Team) . Voltamm3.3.1.Au WE was characterized in phosphate buffer and in phosphate buffer with ferricyanide solutions . The scaWhen the scan direction was reversed towards negative potential the cathodic part of the wave (lower trace) was drown showing the reduction reactions. The phosphate buffer solution (black curve) showed no anodic peaks. The scan direction was reversed towards negative potential at +1,300 mV showing one small cathodic peak at Epc = +503 mV whose origin has not been determined, but is related to some compound/s present in the phosphate buffer. When the phosphate buffer with ferricyanide solution was scanned (red curve) one anodic peak appeared (Epa = +282 mV) corresponding to the oxidation of ferrocyanide to ferricyanide. The scan direction was reversed towards negative potential at +1,500 mV drawing the cathodic part of the wave and two cathodic peaks were seen. One peak around +176 mV corresponding to the reduction of ferricyanide to ferrocyanide and another at +468 mV with a smallest current which has a similar Epc than the phosphate buffer cathodic peak.3.2.La Serenisima brand were performed (M samples (black curve) were scanned only one anodic peak (Epa) appeared around +648 mV (Ipa = 1.65 μA). In M), melamine synthetic milk (0MSM) and one adulterated milk sample (1MSM) voltammograms are plotted. 0MSM (green curve) showed one anodic peak at around Epa of +292 mV (Ipa = 0.51 μA) and one cathodic peak at around Epc = +5.6 mV with a similar current value (Ipc = 0.49 μA) which is not present in M neither in 1MSM.Cyclic voltammetries in milk and adulterated milk samples (with urea or melamine) from erformed  and 3 wiM at Epa = +648 mV is present in 1MSM showing a current reduction (Ipa = 1.06 μA) and it is not present in 0MSM. In M, urea synthetic milk (0USM) and one adulterated milk sample (1USM) voltammograms are plotted. The 0USM voltammogram (green curve) showed one anodic peak at around Epa of +281 mV (Ipa = 0.68 μA) and one cathodic peak at around Epc = +40 mV (Ipc = 0.84 μA) which are not present in M nor in 1USM. In contrast the anodic peak of M is also present in 1MSM showing a current reduction (Ipa = 1.08 μA) but it is not present in 0MSM. From these figures it is noticed that although adulterated milk samples voltammograms have the same shape than M voltammograms, the addition of “synthetic milk” results in a decrease of current values. As the concentration of “synthetic milk” decreased, the greater is the similarity between its cyclic voltammetries and the voltammetries of M.On the other hand, the peak that appeared in versus Ag/AgClsat and performing cyclic voltammetries in milk obtained only one anodic peak located around +732 mV but no cathodic peaks at all. Even though the reacting species present in the samples are not known, in 1991, Jawad et al. [versus Ag/AgClsat and performing cyclic voltammetries in milk postulated that uric acid is the responsible for an anodic peak at +845 mV.Other authors  using a d et al.  using a M and all the adulterations with 0USM and with 0MSM  methods has been applied to original data  and to iM). Sancor) was included in the analysis following the same procedure used for La Serenisima brand. For Sancor samples adulterated milk was represented by an in-between concentration of adulterant (3MSM and 3USM). These adulterated samples are all plotted near La Serenisima adulterated samples and separate from all non-adulterated samples, both La Serenisima and Sancor  analysis to classify milk samples into five different levels of adulteration . A “leave-one-out” strategy was applied to validate the KNN classifier, for non-treated cyclic voltammetries  and for ts level . The lesLa Serenisima milk and then a second batch of non-adulterated and adulterated samples from Sancor was used as a test set. When classification test were performed with first derivative data all the control samples from the test batch (Sancor) were correctly classified as non-adulterated, and none of the adulterated samples was assigned to the control class. All adulterated samples were classified as USM (high or low) irrespectively of the adulteration type.The system was trained with M samples are all correctly classified for both batches. These results suggest that this method is able to discriminate between milk and adulterated milk with “synthetic milk” containing different nitrogen compounds such as urea and melamine reaching a discrimination level of 1% of adulteration. Moreover, the fact that no sample pretreatment is needed, as the addition of extracting solvents, shaking, centrifugation, filtering, diluting, etc. [The ability of the Au WE to discriminate the samples containing “synthetic milk” with urea or melamine is clearly noticed, as ng, etc. –41 is anSince the sad Asian event, more analytical methods are available, as recently reviewed ; most of4.−1) established by WHO to a 75 kg adult it will allow a safe intake of 187.5 mg of melamine per day. Therefore our detection limit is enough to protect an adult whose daily ingest is up to 2.2 liters of milk. These results proves that the screening method presented in this work could provide a simple, rapid and inexpensive method able to detect real cases of milk adulterations and prevent food intoxication with melamine. In addition the urea concentration (down to 121.4 ppm) of milk samples containing “synthetic milk” with urea that were able to discriminate from non-adulterated milk, suggests that this method could be useful to detect urea adulterations given that the maximum concentration of urea allowed for milk is 700 ppm. The performance, low cost, reproducibility and simplicity of the fabricated working electrode and the electrochemical technique demonstrated the advantages of this method to detect adulterated milk samples in a rapid method that could be used to prevent acute intoxications.Best results were obtained with the first derivative of a section of the cyclic voltammetries performed with Au WE in milk samples. f-PCA allowed us to differentiate simulated adulterated milk samples containing urea or melamine from non-adulterated milk samples. We could separate non-adulterated milk from adulterated milk samples with 20, 10, 5, 2 and even 1% of “synthetic milk” with urea or melamine by the correct classification of each adulteration in different groups (levels of adulteration). By this method, the less adulterated samples that were able to discriminated from non-adulterated milk corresponding to adulterated milk with 1% of its total nitrogen from either adulterant (urea or melamine). These samples have a concentration of urea and melamine of 121.4 and 85 ppm, respectively. Considering the maximum melamine concentration found in Chinese adulterated samples  the presented method proves to be useful to detect samples with a concentration 30 times lower that the aforementioned maximum. If we apply the TDI (2.5 mg·kg


It has been repeatedly shown that functional magnetic resonance imaging (fMRI) triggers distress and neuroendocrine response systems. Prior studies have revealed that sympathetic arousal increases, particularly at the beginning of the examination. Against this background it appears likely that those stress reactions during the scanning procedure may influence task performance and neural correlates. However, the question how sympathetic arousal elicited by the scanning procedure itself may act as a potential confounder of fMRI data remains unresolved today. Thirty-seven scanner naive healthy subjects performed a simple cued target detection task. Levels of salivary alpha amylase (sAA), as a biomarker for sympathetic activity, were assessed in samples obtained at several time points during the lab visit. SAA increased two times, immediately prior to scanning and at the end of the scanning procedure. Neural activation related to motor preparation and timing as well as task performance was positively correlated with the first increase. Furthermore, the first sAA increase was associated with task induced deactivation (TID) in frontal and parietal regions. However, these effects were restricted to the first part of the experiment. Consequently, this bias of scanner related sympathetic activation should be considered in future fMRI investigations. It is of particular importance for pharmacological investigations studying adrenergic agents and the comparison of groups with different stress vulnerabilities like patients and controls or adolescents and adults. It has been repeatedly shown that magnetic resonance imaging (MRI) examinations can trigger stress responses Forty-seven, scanner-naive, right handed students from the Technische Universität Dresden were recruited using flyers and public announcements. All subjects had normal or corrected to normal vision. Exclusion criteria included smoking, oral contraceptives, body mass index below 18 or above 26, prior  scanning experience, any history of acute or chronic medical disease or failure to meet MRI compatibility. Subjects received course credit or €10 for participation. All subjects gave their written informed consent. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Technische Universität Dresden. Subjects arrived between 11:00 am and 5:00 pm at the Neuroimaging Center and were asked to come well rested to testing and were also requested not to eat or drink one hour before the experiment. To ensure that all participants had adequate levels of blood glucose they received 2 dl grape juice immediately after the first saliva sample. Six subjects were excluded from further analysis due to response to catch-trials, see below, (n = 2) and incomplete biochemical data (n = 4). Four subjects were additionally excluded from the fMRI analysis due to excessive movement parameters > 3 mm resulting in a total fMRI dataset of n = 37 ; female: n = 6; mean age  =  23.17 (± 3.19)).After entering the lab, subjects were informed about the study protocol and MRI procedure. Then subjects performed a training session of the task for 20 min to control for further learning effects during the fMRI examination. Thereafter subjects were brought into the examination room and prepared for scanning. MRI data were acquired in the following order: functional run 1 (15 min); structural scan (10 min); functional run 2 (15 min). Both functional runs were identical. Saliva samples were obtained before and after the training runs, after the MRI preparation (immediately before entering the scanner) after run 1 and before and after run 2. MRI staff was held constant for all subjects. For a detailed description of the study protocol please see Muehlhan et al., A temporal cued target detection task similar to that used by Coull, et al. (2000) Salivettes® ‘code blue’  with synthetic swabs were used for saliva sampling. Subjects were instructed to gently chew until the swabs were soaked with saliva. During scanning session, subjects lied on the MRI table and were put back in the home position. Swabs were handed to the subject by an assistant to avoid any movement. Samples were stored at –20°C until assayed in the biochemical laboratory. The measurement of sAA activity in the saliva samples was performed as previously described by Muehlhan et al. MRI images were acquired using a 3-Tesla Trio-Tim MRI whole-body scanner . A standard 12 channel head coil and standard headphones were applied. In each functional run, 411 volumes of 38 axial slices with voxel size of 3×3×3 mm (1 mm gap) were acquired sequentially. Each slice had a matrix size of 64×64 voxels resulting in a field of view of 192 mm. Functional measurements were obtained using a T2* weighted gradient echo planar images (EPI) sequence (repetition time (TR) 2200 ms, echo time (TE) 25 ms, flip angle α = 80°). Structural images were obtained by using a Magnetization Prepared Rapid Gradient Echo Imaging (MPRAGE) sequence .A one way ANOVA was performed to calculate changes in sAA over the six sampling points. Subsequent pairwise comparisons were used to identify significant changes between the sampling points. Greenhouse-Geisser adjustments were applied where appropriate. For all significant changes we calculated the percentage sAA change to account for individual variability in initial values. In detail, we calculated the percentage sAA change immediately prior to scanning ((SP3-SP2)/SP2)*100, the percentage sAA change before ((SP5-SP4)/SP4)*100 and during run 2 ((SP6-SP5)/SP5)*100. Since prior results revealed that some but not all subjects showed a sympathetic response in response to the scanner setting Mean reaction times (RT) were calculated for every subject. RTs shorter than 50 ms and longer than 600 ms were counted as errors and were not considered in further analysis FMRI data were analysed using SPM8 . Prior to pre-processing, the first four scans were excluded from analysis to avoid T1 equilibration effects. The remaining functional scans of both runs were spatially realigned and unwarped to correct for interscan movement. Acquisition times were corrected by setting the reference to slice 16 (middle slice). Structural images were registered to the functional scans and all volumes were normalized to the MNI  reference brain by applying a unified tissue segmentation and normalization algorithm. A smoothing kernel of 8 mm full-with half-maximum was used to accommodate interindividual anatomical variability. At the first level regressors were built for the effects of interest  and the effects of no interest  for run 1 and run 2 respectively. All regressors were modelled as single events and time locked to the onset of the cue. A 128 sec. high-pass filter was used to remove non-physiological slow signal shifts. The general linear model was used to calculate regression coefficients  for the regressors and each voxel. For the fixed effects a full factorial design was used to calculate the task induced activation and deactivation (run 1 and run 2). In the next step one sample t-tests for run 1 and run 2 were calculated for the main effect of the task and the percentage sAA change was integrated as a covariate. To identify brain regions that were influenced by sympathetic changes during the task, conjunction-null analyses ANOVA results indicated significant changes of sAA during the six sampling points . Subsequent pairwise comparisons revealed no significant sAA difference before and after the training session outside the scanner  indicating no sympathetic activation that could be attributed to the task performance itself. Immediately prior to scanning, however, sAA rose significantly , up to its peak: SP3 > SP2; SP5; SP6; p<0.05 and remained at high levels during run 1: SP3, SP4: 0.286. Then a significant decrease was observed while the structural images were acquired (SP4 > SP5: p = 0.002) followed by a second increase at the end of the examination  see . We furtF   =  3.694, p = 0.030, η = 0.101]. Subsequent pairwise comparisons yielded significant differences between the training session and the both runs (run1 and run2) p<0.05. RTs of run 1 and run 2, however, did not differ significantly p = 0.660.   = 7.404, p<0.001, η = 0.188]. Subsequent pairwise comparisons showed that subjects performed the task with the highest accuracy during the training phase. After entering the scanner, accuracy rates decreased significantly T0 > R1, R2, p  =  but did not differ between run 1 and run 2  are presented in Reaction times (RT) negatively correlated with task induced activation in the left SMA: r = –.400, p<0.014, and with TID in the left angular gyrus r = –.410, p<0.012. In other words faster reaction times were associated with higher SMA activation and a lower suppression of left angular gyrus activity. Positive correlations could be observed between correct responses and task induced activation in the following structures: left SMA: r = .403, p<0.013, left lentiform nucleus: r = .374, p<0.022 and the right declive: r = .395, p<0.016. Scatter plots and coefficients of determination  were known key nodes of the default mode network (DMN) that was supressed during active task performance In addition, the positive association between the reported regions and sympathetic reactions corroborate findings from prior studies that showed associations between sympathetic arousal and task independent processes. After a median spilt procedure of high and low skin conductance responses (SCR) during a cognitive challenging task Zang and colleagues Results, however, have to be interpreted in the methodological limits of the investigation. We cannot offer a direct control condition where subjects are subdivided either to a lab or a scanner session; we can nevertheless draw some within-subject conclusions based on the course of the entire experiment that included a 20 minute lab training prior to the scanner session. During this training no significant changes in sAA were found. SAA peaked for the first time immediately prior to scanning. This finding was supported by other studies that recorded sympathetic Furthermore, only scanner naive subjects were included in the present study, it is possible that subjects habituate to the scanner environment after repeated participation in fMRI investigations which in turn prevented the observed behavioural and neural effects. However, in another study conducted in our lab, we were able to show that repeated fMRI scanning led to a habituation of subjective distress but increased the number of subjects showing a neuroendocrine response Furthermore, we here used a very simple target detection task. Other functions like attentional inhibition Finally, it should be also taken into account that the observed effects may vary due to the population studied . Differences in sympathetic activity between groups could be misinterpreted as specific correlates of a disease or age related effects. Future studies are clearly needed to evaluate if patients and controls or different groups differ in sympathetic activation during fMRI examinations.We here demonstrated that sympathetic stress reactions correlate with behavioural data and neural activation patterns. These results are in line with prior findings but showed for the first time that sympathetic arousal elicited by the scanning session itself could account for such effects. It could be assumed that studies investigating adrenergic agents or patient groups are affected by this bias of scanner related arousal. As a consequence of high sympathetic arousal at the beginning of the scanning procedure, it might be advantageous to acclimatise the subjects to the scanner setting. Ten to 15 minutes last out in order to decrease sympathetic and subjective stress responses


ZAR1, which has been reported to be aberrantly methylated in a few human cancers.Hepatocellular carcinoma (HCC) is one of the most common human malignancies in the world, and its prognosis is generally poor. Epigenetic alteration such as DNA methylation has been shown to be important in the development of human cancers including HCC. Here, we analyzed the methylation status of ZAR1 in 88 HCV-positive HCC and matched nontumorous liver tissue samples and 4 normal liver tissue samples used as a control using MassARRAY EpiTYPER. Further statistical analysis was performed to determine the relationship between methylation level and patient clinicopathological features and prognosis.We investigated the methylation status of ZAR1 exon 1 showed a higher methylation level in all 88 HCC than in nontumorous tissues. The hypermethylation group, whose cancer tissues showed a twofold or higher methylation level compared with nontumorous tissues, showed a significantly higher serum AFP (p = 0.018) and lower serum albumin (p = 0.001) and single rather than multiple tumors (p = 0.031) compared with the hypomethylation group. Multivariate regression analyses were performed to identify which of the following factors were the predictors of the hypermethylation group: serum albumin, AFP, and tumor multiplicity. This study showed that patients who had Zar1 hypermethylation in the HCC tissues had a significantly lower serum albumin level than those in the hypomethylation group (p = 0.007).CpG islands in ZAR1 hypermethylation affects HCC development, it could be a potential marker to detect HCV-related HCC.Although it is still unknown how  Despite the fact that diagnosis and treatment have been established for HCC, the prognosis is generally poor. Even after liver resection, 25%, 50%, and 80% of HCC patients suffer relapses within 1, 2, and 5 years, respectively. Although the incidence of nonBnonC-HCC has recently shown an increasing trend, there still are many HCV-positive HCC patients in Japan  was analyzed in 88 HCV-positive HCC and 4 normal liver tissues, which were circumjacent to colon cancer liver metastasis. The average methylation level of all CpG sites in Zar1 in each individual was calculated, and 67 of 88 cases showed a significantly higher methylation level in HCC than in nontumorous tissue (p < 0.05) . Because it is already known that HCV-positive nontumorous tissues undergo some epigenetic changes, we also compared the methylation level of Zar1 in normal liver tissues with that in HCV-positive nontumorous tissues; however, no difference was found between the tissues (data not shown).The methylation level of CpG islands in ZAR1 compared with nontumorous liver tissues. A total of 22 and 66 patients were enrolled in hypermethylation hypomethylation groups, respectively. When the background clinicopathological features of the hypermethylation and hypomethylation groups were compared using univariate analysis, there were significant differences in levels of serum AFP (p = 0.018) and albumin (p = 0.001) and tumor multiplicity (p = 0.031) (Table p = 0.007)  finger in its C-terminus. PHDs are found in two major classes of proteins, such as transcriptional activators, repressors, or cofactors and subunits of complexes that modulate chromatin  and lower level of serum albumin (p = 0.001) compared with the hypomethylation group, using univariate analysis. In addition, all the patients in the hypermethylation group developed only single, but not multiple, tumors (p = 0.031). Multivariate regression analyses were performed to identify the predictors of the hypermethylation group. The patient with low serum albumin level had an increased risk for hypermethylation (p = 0.007). In general, it was already known that a high serum AFP level and low serum albumin level are associated with development and poor prognosis of HCC . For these reasons, it is suggested that ZAR1 hypermethylation could be a novel and good marker to detect HCC and also could be a potential marker for HCC prognosis.ZAR1 mRNA expression was not detected in any of the tissue specimens in this study. There is a possible explanation for this result. The aberrant hypermethylation of ZAR1 may be the result of a secondary effect of the tumorigenesis process, or the methylation level of Zar1 exon 1 may be involved in the regulation of other genes distant from Zar1. Further studies are needed to understand the role of hypermethylation of the Zar1 genomic region in HCC development.Zar1 genomic region is higher in HCV-related HCC than in nontumorous tissues and that patients with Zar1 hypermethylation had a significantly lower serum albumin level than those from the hypomethylation group. Although it is still unknown how ZAR1 hypermethylation affects HCC development, it could be a marker for HCV-related HCC and also could be a potential marker for predicting HCC prognosis.This study showed that the methylation level of the Eighty-eight hepatitis C virus-positive HCC and matched nontumorous tissue and four normal liver tissues, which were circumjacent to tissues of metastatic liver cancer, were obtained from the Nihon University Hospital, Japan, from 1995 to 2010. Informed consent was obtained from all patients. All liver tissue specimens were immediately frozen in liquid nitrogen and stored at −80°C until use. Nontumorous tissues were excised with sufficient margins from the cancer. This study was approved by the Institutional Review Boards of Nihon University School of Medicine.DNA was extracted using the standard phenol–chloroform purification method or a QIAamp DNA Mini Kit . Bisulfite modification of DNA was performed using an EZ DNA Methylation Gold Kit . Bisulfite-treated genomic DNA was amplified using HotStar Taq Polymerase (Qiagen) for 15 min at 94°C followed by 45 cycles of 20 s at 94°C, 30 s at 56°C, and 1 min at 72°C, with a 3-min final extraction at 72°C. The polymerase chain reaction (PCR) products obtained were subjected to gel electrophoresis and then measured using Sequenom MassARRAY.ZAR1 primer was designed using Methyl Primer Express® Software to span the closely adjacent DMRs or the GC rich region, as indicated. The primers used for the quantitative ZAR1 methylation analysis were designed to anneal to the 374-bp bisulfite-treated fragment for the MassARRAY EpiTYPER . The reverse primer has a T7-promoter tag for in vitro transcription (5′-cagtaatacgactcactatagggagaaggct-3′), and the forward primer is tagged with a decamer to balance the temperature (Tm) (5'-aggaagagag-3′). These primers were purchased from Operon . PCR amplification was performed using HotStar Taq Polymerase (Qiagen) in a 5-μl reaction volume using PCR primer at a final concentration of 200 nM and 1 μl of bisulfite-treated DNA (~20 ng/ml). After treatment with shrimp alkaline phosphatase, 2-μl aliquots of the PCR products were used as a template for in vitro transcription and RNase A cleavage for the T-reverse reaction, as described in the manufacturer’s instructions (Sequenom hMC). The samples were desalted and spotted on a 384-pad SpectroCHIP (Sequenom) using a MassARRAY nanodispenser  followed by spectral acquisition on a MassARRAY Analyzer Compact MALDI-TOF MS (Sequenom). The resultant methylation calls were analyzed using EpiTYPER software v1.0 (Sequenom) to generate quantitative measurements for each CpG site or an aggregate of multiple CpG sites. A standard curve of quantitative DNA methylation analysis was constructed using 0%, 50%, and 100% methylated samples. The amplified DNA was not methylated at all in any of the CpG sites and was used as unmethylated (0%) control. M.SssI double-treated DNA was prepared according to the manufacture’s protocols  at 37°C for 1 h followed by inactivation at 65°C for 20 min for 100% methylation samples. By mixing equal amounts of 0% and 100% control samples, the 50% methylation samples were prepared. The standard curve was fitted, and methylation levels were adjusted and quantified.A Sequenom MassARRAY quantitative DNA methylation analysis was performed . Differences were considered significant at p < 0.05.Comparison of clinicopathological features between the hypermethylation and hypomethylation groups was analyzed by Fisher’s exact test or Mann–Whitney


Mitochondria are known to be a major source and target of oxidative stress. Oxidative stress increases during aging and is suggested to underlie in part the aging process. We have previously documented an increase in endogenous caspase-2 (casp2) activity in hepatocytes obtained from old (28 months) vs. young mice (5 months). More recently, we have shown that casp2 is activated by oxidative stress and is critical for mitochondrial oxidative stress-induced apoptosis. Since casp2 appears integral to mitochondrial oxidative stress-induced apoptosis, in this study we determined whether loss of casp2 altered the production of mitochondrial reactive oxygen radicals (mROS) as a function of age in intact living hepatocytes. To stimulate mitochondrial metabolic activity, we added a mixture of pyruvate and glutamate to hepatocytes while continuously monitoring endogenous mROS production in the presence or absence of rotenone and/or antimycin A. Our data demonstrate that mROS production and neutralization are compromised in hepatocytes of old mice. Interestingly, casp2 deficient hepatocytes from middle age mice (12 months) had similar mROS neutralization kinetics to those of hepatocytes from old WT mice. Rotenone had no effect on mROS metabolism, whereas antimycin A significantly altered mROS production and metabolism in an age-dependent fashion. Our results indicate that: (1) hepatocytes from young and old mice respond differently to dysfunction of the mitochondrial electron transport chain; (2) age-dependent alterations in mROS metabolism are likely regulated by complex III; and (3) absence of casp2 accelerates age-dependent changes in terms of pyruvate/glutamate-induced mROS metabolism. With aging, there is a general decrease in liver size and thus in hepatocyte number  was obtained from Molecular Probes.Casp2/−− mice were a gift from Dr. Carol Troy. Only male mice were used in these studies. In the present studies, we used mice at ages 5, 12 and 28 months of age. This represents for 5 month old WT mice 12 % of their maximum lifespan, 73.9 % for 28 month old WT, and 28.7 % and 31.7 % for 12 month old WT and Casp2/−− respectively.Casp2/−− and WT mice. In brief, animals were anesthetized and their livers perfused with basic medium solution containing 0.5 mM EGTA  followed by a collagenase solution containing 1 mM CaCl2. After washing and dispersing the cells, they were counted. An average of 25 million hepatocytes per liver was obtained.Hepatocytes were isolated using the method of Herman et al. Casp2/−− and WT mice. After hepatocytes were isolated as explained above, cells were washed and dispersed, and counted. After determining cell viability (85–95 %), hepatocytes were seeded at 250,000 cells per well in two-well glass chambers and incubated for a minimum of 9 h to a maximum of 20 h. Generation of ROS over time was monitored with MitoSOX as previously described  was added to the chamber to a final concentration of 5 mM. In some experiments, hepatocytes were treated with 20 μM of rotenone, 20 μM antimycin A or 20 μM of a mixture of both and cells were incubated for 30 min. Cells were then washed with PBS and prepared as described above for mROS measurements. Three regions of interest were independently scanned and each experiment was repeated from six to nine times. Data was normalized for basal intensity by first dividing the intensity obtained at each time point by the initial intensity. Then, the best fit curve in nonlinear regression was found and used to normalize each time point. Analysis was performed with GraphPad Prism software, version 5. For specific time points, one-way ANOVA was used to analyze statistical differences, followed by Bonferroni posttests to discern what groups where significant. At least three animals were used for each sampling group. For the untreated experiments, results were pooled together and statistically analyzed. The results provided for hepatocytes treated with mitochondrial complex inhibitors are presented as individual graphs to highlight the similar response pattern.Animals were handled according to the University’s Institutional Animal Care and Use Committee approved regulations, protocols, and standards. Hepatocytes were isolated from 5 to 28 month old WT mice or 12 month old Casp2/−− hepatocytes isolated from middle age mice and compared it to the response of hepatocytes obtained from age-matched WT mice  there is a difference between the way hepatocytes from young and old mice respond to dysfunction of the mitochondrial electron transport chain and subsequent production and neutralization of mROS; (2) this difference is most probably due to age dependent dysfunction of complex III; and (3) absence of casp2 in hepatocytes obtained from middle age mice respond in a similar way as hepatocytes obtained from old WT mice in terms of PG-induced mROS metabolism.Our previous studies have shown a progressive increase in the activity of caspases that mediate apoptosis induced through the mitochondrial pathway as liver ages  All our previously published data on casp2 deficient mice biology has been obtained using male mice; and (2) hepatocytes used in these experiments were cultured in vitro with specific media, thus removing any potential impact of gender in the results. Hepatocytes are cells with a high degree of metabolic activity and are greatly enriched in mitochondria, which in turn leads to production of high levels of ROS. We examined ROS production from mitochondria in hepatocytes isolated from young (5 months), middle age (12 months) and old (25 months) WT mice. Our data demonstrated that hepatocytes of old mice do not produce as much initial mROS as hepatocytes from young mice when mitochondrial respiration is accelerated. In addition, the kinetics of mROS neutralization were not as efficient in hepatocytes isolated from old mice compared to that observed in hepatocytes isolated from young mice. These results indicate that older hepatocytes lack the metabolic efficiency found in younger hepatocytes.Casp2/−− mice display a greater difficulty in neutralizing mROS than hepatocytes from their age-matched WT counterparts, and in fact resemble the characteristics of mROS neutralization found in hepatocytes obtained from old WT mice. These data implicate casp2 in the preferential metabolism of mROS generated from complex III. Recent evidence suggests that when complex III is inhibited, complex II may be a source of ROS   in liver cell energy metabolism (Ramanujan and Herman Casp2/−− mice (Lopez-Cruzan et al. Casp2/−− mice have increased cellular levels of oxidized proteins, lipid peroxides and DNA damage. Fibroblasts and neurons from Casp2 null mice generate higher levels of endogenous ROS that is associated with decreased levels of antioxidant enzymes (Tiwari et al. 2O2 detoxifiers. In the absence of casp2, these antioxidants are decreased, and in the case of MnSOD, it might explain the increased mROS production seen in older WT and middle age casp2 null hepatocytes. Taken together, these observations demonstrate strong mechanistic links between casp2, oxidative stress and aging.Lastly, a recent publication (Shalini et al.


The lateral prefrontal and orbitofrontal cortices have both been implicated in emotion regulation, but their distinct roles in regulation of negative emotion remain poorly understood. To address this issue we enrolled 58 participants in an fMRI study in which participants were instructed to reappraise both negative and neutral stimuli. This design allowed us to separately study activations reflecting cognitive processes associated with reappraisal in general and activations specifically related to reappraisal of negative emotion. Our results confirmed that both the dorsolateral prefrontal cortex (DLPFC) and the lateral orbitofrontal cortex (OFC) contribute to emotion regulation through reappraisal. However, activity in the DLPFC was related to reappraisal independently of whether negative or neutral stimuli were reappraised, whereas the lateral OFC was uniquely related to reappraisal of negative stimuli. We suggest that relative to the lateral OFC, the DLPFC serves a more general role in emotion regulation, perhaps by reflecting the cognitive demand that is inherent to the regulation task. The capacity to regulate the generation, experience and expression of emotion is a central aspect of mental health per se and activations reflecting the recruitment of networks that are shared between different cognitive regulation processes more generally. Although previous research confirms that such a general network of cognitive control Instruction-induced reappraisal of an emotionally salient stimulus is the most extensively studied form of cognitive emotion regulation. During reappraisal, cognitive strategies are used to reinterpret the meaning of an emotion-eliciting stimulus in terms of a non-emotional event thereby attenuating automatic emotional responses Although there are other candidate regions with relevance for emotion regulation, such as the subgenual anterior cingulate cortex The protocol was approved by the ethics committee of Karolinska Institutet.We recruited 61 participants who were free from self-reported life-time psychiatric or neurological disease and medication. Prior to analysis, we excluded 2 left handed participants and 1 participant with abnormal brain anatomy leaving a final sample of 58 (26 men) right-handed, participants with a mean age of 24.02 years (SD = 2.26). All participants gave informed consent and were paid 400 SEK (approximately 55 USD) for their participation. Due to technical problems, the behavioral rating data were missing for 2 participants, who therefore were excluded from the statistical analyses of the rating data.t-tests yielded significantly lower valence (p<.05 ) and higher arousal (p<.05 ) ratings for negative compared to neutral pictures, whereas there were no differences in arousal or valence between the two negative picture sets (p>.05) or between the two neutral picture sets (p>.05)  .05) see . Stimuliwww.magconcept.com).Most previous studies on instruction-induced emotion regulation have typically excluded a control condition involving a regulation instruction preceding neutral picture trials. The basic rationale for this was described in an early emotional suppression study An anatomical scan and fMRI data were obtained using a GE Sigma Echo Speed 1.5 T scanner and an 8-channel head-coil. Functional scans were obtained using a gradient echo sequence T2*-weighted echoplanar imaging (EPI) scan  and a field of view (FOV) = 22 cm×22 cm, matrix size = 64×64). The first ten scans were defined as dummy scans to allow for longitudinal T1-equilibrization and these were not included in the analysis. Functional image acquisition was divided into 2 runs of 245 volumes with a break of approximately 20–30 sec between the runs. A T1-weighted structural image (3D-SPGR) was also collected at the end of the experiment.All imaging data were analyzed with SPM8  running on Matlab20010a . For each subject, individual images were first slice-time corrected to adjust for acquisition time differences between the slices and realigned to the first volume to correct for head movement. The T1-weighted image was then co-registered with the mean EPI image and subsequently segmented using the unified segmentation approach as implemented in the “New Segment” routine in SPM8 to generate gray and white matter images. These images were then used within the DARTEL toolbox to create structural templates across subjects as well as individual flow fields that were then used to spatially normalize the EPI images into MNI space. Finally, all images were spatially smoothed with a Gaussian kernel with a full width at half maximum (FWHM) of 8 mm and temporally high-pass filtered with a cutoff of 128 to remove low-frequency drifts.Statistical analysis was performed using standard procedures for fMRI involving a fixed effect model at the single-subject level and a random effects model at the group level p<.05, FWE together with a 10 voxel cluster threshold. To more fully explore the neural regions related to regulation, we supplemented these ROI analyses with whole brain analyses that were considered significant at p<.05, FWE corrected, together with a cluster threshold of 10 voxels (see Supplementary data). All reported coordinates refer to Montreal Neurological Institute (MNI) space.Given the role of the amygdala in emotional processing F = 83.55, p<.001, η2 = .60 and Valence F = 194.31, p<.001, η2 = .78, as well as a significant Instruction × Valence interaction F = 60.22, p<.001, η2 = .52. The reliable interaction was driven by significantly lower discomfort ratings on Reappraise  than on Attend  trials. To confirm this interpretation, we first subtracted ratings during the viewing of neutral pictures from ratings during the negative pictures for each instruction and then compared these difference terms, which resulted in a highly significant difference, p<.001 , p<.05, (FWE). More specifically, we wanted to confirm that we (a), accomplished successful emotion elicitation, as demonstrated by greater amygdala activation in the contrast Attend negative > Attend neutral T = 4.24, kE = 192 voxels; left amygdala x = −20, y = 1, z = −18, T = 4.80, kE = 192 voxels) as well as during the Attend negative > Reappraise negative condition , p<.05, (FWE).First, to confirm that our experimental task effectively engaged the amygdala when participants were exposed to negative pictures, we looked at the main effect of Valence within the predefined amygdala ROI. As expected, the analysis revealed a significant bilateral amygdala activation in response to negative compared to neutral pictures  or neutral stimuli , the lateral OFC was uniquely activated during reappraisal of negative stimuli . To confp<.05, FWE).For clarity, we also confirmed that neither of our prefrontal ROIs was significantly more activated during reappraisal of neutral trials as compared to during the reappraisal of negative trials. Neither ROI showed significant activation in Reappraise neutral > Reappraise negative  the DLPFC and the lateral OFC both contribute to negative emotion regulation, but (b) that these regions partly reflect different aspects of the emotion regulation process. Critically, our results showed that in contrast to the more general pattern of activation in the DLPFC, the lateral OFC was specifically and uniquely activated during reappraisal of negatively valenced stimuli. These findings add to a growing literature focused on disentangling the contributions of different prefrontal regions during emotion regulation.Table S1Whole-brain activations for the main effect of Instruction. BA = Brodmann area; R = Right; L = Left. Coordinates: MNI system. All reported activations are significant at p<.05 (FWE).(DOC)Click here for additional data file.Table S2Whole-brain activations for the effect of Instruction × Valence interaction. BA = Brodmann area; R = Right; L = Left. Coordinates: MNI system. All reported activations are significant at p<.05 (FWE).(DOC)Click here for additional data file.Table S3Whole-brain activations for “Reappraise” vs. “Attend” during negative and neutral trials. BA = Brodmann area; R = Right; L = Left. Coordinates: MNI system. All reported activations are significant at p<.05 (FWE).(DOC)Click here for additional data file.Table S4Whole brain activations in the conjunction analysis of Reappraise negative > Attend negative and Reappraise neutral > Attend neutral. BA = Brodmann area; R = Right; L = Left. Coordinates: MNI system. All reported activations are significant at p<.05 (FWE).(DOC)Click here for additional data file.


Hall mark in Alzheimer’s Disease (AD) is the aggregation of amyloid-β (Aβ) peptide into oligomers and fibrils. Nowadays, the soluble oligomers are believed to be the most neurotoxic species, probably the causative agents in AD. Amyloid-beta fibrils on the other hand may serve as reservoirs for small toxic oligomers. While it is well known from experiment, that the aggregation process is modulated by salt concentration in solution, the molecular details of the underlying interactions are not.Salts occur ubiquitously in physiological environments and are known to have profound effects on the solubility of proteins (Hofmeister series). Monovalent alkali metal ions exhibit a more subtle effect on Aβ aggregation in experiment than doubly charged species ,2.In this contribution we investigate the presence of the so-called ‘sodium-effect’ in fibrillar Aβ oligomers. This effect was originally described for dendrimer micelles formation modulating the self-organization of amphiphilic carboxylates: Na+ forms bridging complexes with carboxylate groups, in contrast to K+ .Molecular dynamics simulations for a systematic series of single and double layer fibrillar Aβ oligomers in aqueous 150mM salt solution provide insights about the stabilizing interactions between the cations and charged Aβ key residues (e.g. Glu22). Comparison with a previous computational study at low ion concentration  show sim


We employed fluorescence molecular tomographic (FMT) noninvasive imaging of these molecular activities using cathepsin sensing  and αvβ3 integrin (IntegriSense) near-infrared fluorescence (NIRF) agents. A statistically significant increase in the ProSense and IntegriSense signal was observed within the chest region of apoE−/− mice (P < 0.05) versus C57BL/6 mice starting 25 and 22 weeks on high cholesterol diet, respectively. In a treatment study using ezetimibe (7 mg/kg), there was a statistically significant reduction in the ProSense and CatB FAST chest signal of treated (P < 0.05) versus untreated apoE−/− mice at 31 and 21 weeks on high cholesterol diet, respectively. The signal of ProSense and CatB FAST correlated with macrophage counts and was found associated with inflammatory cells by fluorescence microscopy and flow cytometry of cells dissociated from aortas. This report demonstrates that cathepsin and αvβ3 integrin NIRF agents can be used as molecular imaging biomarkers for longitudinal detection of atherosclerosis, and cathepsin agents can monitor anti-inflammatory effects of ezetimibe with applications in preclinical testing of therapeutics and potentially for early diagnosis of atherosclerosis in patients.Inflammation as a core pathological event of atherosclerotic lesions is associated with the secretion of cathepsin proteases and the expression of  Atherosclerosis, a progressive inflammatory disease, results in the development of plaques within the arterial walls that may contain intracellular and extracellular lipids, a variety of inflammatory cells, extracellular matrix, smooth muscle cells, fibroblasts, and calcification –4. Acuteαvβ3 integrin  cholesterol diet which included 9% of their caloric intake from fat  were used as an animal model of atherosclerosis. In the longitudinal study, C57BL/6 mice  fed on a normal chow diet served as controls. In the treatment study, apoE−/− mice were fed the diet described above with or without cholesterol absorption inhibitor ezetimibe (7 mg/kg/d). All experiments were approved by our Institutional Animal Care and Use Committee (IACUC).Ten-week old apoE−/− mice (8 to 10 months on diet), which developed atherosclerotic lesions, were injected with ProSense 750 , ProSense FAST 750 , or IntegriSense 750  via tail vein 24 hr or 48 hr before FMT imaging, respectively. Images were acquired by FMT2500 (Perkin Elmer) at 750 nm wavelength. The resulting 3D dataset was reconstructed and the region of interest (ROI) was defined within the chest area  were then acquired with the FMT2500. Aged apoEest area  using thin vivo FMT signal noninvasively, the mice were imaged with a GE Explore Locus Ultra  after FMT acquisition using a CT bed designed to hold the FMT animal cassette therefore ensuring the animal remained in the same position. The FMT dataset was exported in Dicom format and then co-registered to the CT dataset using Amira 5.2 .To verify the μm cell strainer  and washed with PBS. The blood samples were incubated in ACK buffer  to remove blood cells and then washed with PBS. For multicolor flow cytometry analysis of lymphoid cells, myeloid cells (including monocytes/macrophages and neutrophils) and other cells , cells were incubated with a cocktail of mAbs  against T cells (CD90-FITC),B cells (B220-FITC), NK cells , and myeloid cells (CD11b-PE) at 4°C for 30 min and then analyzed on an LSRII . Acquisition data was analyzed by the FlowJo software .After probe-injected mice were euthanized, their aortas were excised and terminal blood samples were collected. The aortas were minced with fine scissors and dissociated into single cells by incubating with Liberase TH and TM  at 37°C for 1.5 hours. The resulting cell suspensions were then passed through 40 μg/mL; MCA-1957, AbD Serotec, Raleigh, NC) for 1 hr at room temperature. Sections were washed in PBS and incubated with AlexaFluor 488 goat anti-rat IgG  for 1 hr and again washed in PBS. Sections were mounted using a fluorescent mount medium with or without DAPI counterstain and examined with a Nikon E-1000 microscope equipped with the appropriate band-pass filters.To demonstrate co-localization of macrophages and the IntegriSense 680 and ProSense 680 probes, selected vessels were stained with a monoclonal anti-CD68 antibody. Ten micron-thick cryosections were prepared and fixed in ice-cold acetone/ethanol (3 : 1) for 10 min. and washed in PBS. Nonspecific binding was blocked by incubation with 5.0% nonimmune goat serum for 30 min followed by incubation with the rat anti-mouse CD68 antibody (5 −/− (n = 15 for each agent) and C57BL/6  mice were injected with ProSense750 or IntegriSense750 and imaged with the FMT2500 as described above. The scans were conducted at the times indicated in the figures.For the longitudinal study, both ApoE−/− mice  via tail vein 24 hr before imaging. IntegriSense  injected mice were images 48 hr after injection. The imaging acquisition was carried out at the time points as depicted in Figures For the treatment study, ProSense, CatB FAST agent or ProSense FAST  were injected into apoE−/− mice were euthanized, their aortas were excised and fixed in 10% buffered formalin . Slides were subsequently incubated with a peroxidase-conjugated anti-mouse IgG antibody and 3′3′-diaminobenzidine (DAB) chromogen to identify macrophages within the plaque regions and counterstained with hematoxylin  were collected at the end of the treatment study, and the HDL-C and total cholesterol levels were determined by following the manufacturer's protocol of HDL-Cholesterol E kit  with and without adding precipitating reagent, respectively. Terminal serum samples (n = 12) were collected at the end of the treatment study and assayed for and ezetimibe-glucuronide using Aria LC-MS/MS with a standard calibration range from 5 to 10,000 nM and a quantification limit of 5 nM. Terminal plasma samples  (αvβ3 integrin was determined to be ~ 0.5 nM [αvβ3 (The g (NIRF) . The aff~ 0.5 nM  and the [αvβ3 . in vivo FMT analysis, we compared fluorescent signal within the ROI drawn to capture the entire volume of the chest region with the signal measured within the ROI drawn on the excised arteries (for details see Methods) to ensure the accuracy of the in vivo quantification  measured ex vivo for all evaluated agents  and Cat B FAST , but not for IntegriSense   [The r growth  and RGD-clerosis –42. We e–950 nm) , 30, witin vivo detection of molecular imaging biomarkers in both apoE−/− and control C57BL/6 mice and confirmed the localization of the measured fluorescence using CT. We found that the FMT measured concentrations of imaging biomarkers in the thoracic area of apoE−/− mice indicate that these agents accumulated in the aortic arch and the roots of adjacent carotid and subclavian arteries  similar to previous reports [in vivo measured agent concentrations of both cathepsin-activated and αvβ3 integrin targeted agents correlated well with the ex vivo fluorescence in the same areas of the dissected arteries of apoE−/− mice. We employed FMT technology for the quantitative  reports . Moreoveαvβ3 integrin [αvβ3 integrin-dependent angiogenesis, originating from adventitial vasa vasorum and resulting in immature, fragile, and leaky vasculature, triggering intraplaque hemorrhage [−/− mice and showed that cathepsin agents were activated primarily in myeloid (monocytes and macrophages) cells while the αvβ3 integrin agent was found not only in myeloid cells but also in lymphoid and stromal  cells indicating that IntegriSense likely detects inflammatory cells, angiogenesis, and cell proliferation. Flow cytometry of blood from animals injected with cathepsin agents detected very low levels (compared to aortas) of the agents in both myeloid and lymphoid cells 24 hours after agent injection, while αvβ3 agent was also detected in both cell types, albeit at much higher levels for myeloid cells, indicating again involvement of αvβ3 integrin in the development and proliferation of inflammatory cells.An early stage of atherosclerotic lesions is characterized by infiltration of monocytes and macrophages and their development into foam cells –3 controintegrin . As the integrin –22, 24. morrhage , 26. To αvβ3 agent was found in advanced atherosclerotic lesions densely distributed in the adventitia and some was also co-localized with macrophages within the core of the lesions reflecting expected development of new vasculature and already discussed expression on the inflammatory cells. FMT measured concentrations of cathepsin agents ProSense and Cat B FAST in apoE−/− mice correlated well with the number of macrophages detected in the six different areas of the aortic arch, supporting the flow cytometric and immunohistochemistry data. However, the correlation between the macrophage count and FMT measured concentrations of αvβ3 agent was poor, which was expected giving the high levels of αvβ3 on the other cells types within the plaque. Immunofluorescence detected the activated ProSense agent in the center of the atherosclerotic lesions, some of it co-localized with resident macrophages confirming that cathepsins are activated in the inflammatory cells and later released from the dead cells into the core of the plaque. The −/− mice and human patients has been documented many times [−/− mice can therefore potentially serve as an imaging biomarker of inflammation. Co-localization of cathepsins  and macrophages in the core of atherosclerotic lesions of apoEny times , 34, 35,ny times  polymer-αvβ3 integrin agent with macrophages and its localization in the adventitia agree well with the previously documented role of αvβ3 integrin in the transformation of monocytes and macrophages in the core of atherosclerotic lesions [αvβ3 integrin agent is therefore, contrary to cathepsin agents, a molecular imaging biomarker of both inflammation and angiogenesis. The poor correlation between the FMT measured αvβ3 agent concentrations and the macrophage counts may indicate binding of this agent to cells other than inflammatory cells within the atherosclerotic lesions. Binding to myeloid cells within the blood may also influence the FMT signal.The co-localization of  lesions , 39–42 a lesions , 42, 43.−/− mice on regular chow and even greater in mice on chow containing elevated levels of fat and/or cholesterol translate into the formation of atherosclerotic lesions in the aortic arch and proximal arteries as early as 3 months after the birth [in vivo measurements of apoE−/− mice fed with a high cholesterol diet found, when compared to C57BLK/6 mice fed with regular chow, elevated levels of the cathepsin probe activation as early as 15–20 weeks after diet initiation. The levels of activation started to grow exponentially after 25 weeks with an increasing variability among individual animals. In a similar set of FMT measurements, the accumulation of αvβ3 agent in atherosclerotic lesion of apoE−/− mice was detected two to five weeks later and then the concentration of the agent was again increasing exponentially. Our data indicate a good correlation between the activation of cathepsin agents and atherosclerosis-associated inflammatory processes in a longitudinal study using these agents which may provide a noninvasive early means of detection of inflammation in developing atherosclerotic lesions of apoE−/− mice. The somewhat delayed onset of the FMT measured αvβ3 agent accumulation might be the result of the later onset of plaque associated angiogenesis. Since both elevated inflammation as well as angiogenesis are characteristics of vulnerable plaques, both cathepsin as well as αvβ3 integrin agents may serve in their in vivo detection. Elevated levels of LDL in the blood of apoEhe birth , 45, 47.−/− mice fed on high cholesterol diet. However, to our surprise, the new and nonselective fast acting agent ProSense FAST showed a statistically significant difference only at the 12 week time point, while the polymer-based ProSense detected the statistically different inhibition only at the 31-week time point. The most sensitive for the detection of inhibition by ezetimibe was Cat B FAST, finding the statistically significant inhibition at both 21 and 31 weeks. This may indicate that the selectivity rather than the size of the agent, as might be concluded from previous studies [αvβ3 integrin selective agent did not detect ezetimibe-induced inhibition of atherosclerosis progression in apoE−/− mice. This is likely a reflection of a broader spectrum of processes involved in the accumulation of αvβ3 agent in the atherosclerotic lesions. Some of these processes may not be affected by the treatment with ezetimibe or they are affected on different time scales. Hence, a more detailed analysis of the functions of these two types of imaging biomarkers may contribute new details on the development of atherosclerotic lesions. Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, has been shown to reduce plasma LDL, increase HDL, and inhibit atherosclerosis associated inflammation , 45, 48. studies , is critin vivo imaging evaluation of new cathepsin activatable and αvβ3 integrin targeted agents, prepared using requirements for the design of new therapeutics. Using FMT, we measured quantitative changes in atherosclerosis-associated inflammation in apoE−/− mice and compared these to the previously studied polylysine-based cathepsin agent. We have demonstrated the importance of cathepsin agent selectivity and capability of all agents to quantitatively detect longitudinal progress in inflammation and angiogenesis of atherosclerotic lesions. We have further showed that cathepsin agents detect inhibition of inflammation as a result of prophylactic treatment with the anti-inflammatory therapeutic, ezetimibe [Here we describe the functional zetimibe , 45.