Campylobacter jejuni, a leading cause of bacterial gastroenteritis. Here, we demonstrate comprehensive mapping of the O‐glycosylation of flagellin from Campylobacter jejuni 11168 by use of a bottom‐up proteomics approach that incorporates differential ion mobility spectrometry  together with proteolysis with proteinase K. Proteinase K provides complementary sequence coverage to that achieved following trypsin proteolysis. The use of FAIMS increased the number of glycopeptides identified. Novel glycans for this strain were identified (pseudaminic acid and either acetamidino pseudaminic acid or legionaminic acid), as were novel glycosylation sites: Thr208, Ser343, Ser348, Ser349, Ser395, Ser398, Ser423, Ser433, Ser436, Ser445, Ser448, Ser451, Ser452, Ser454, Ser457 and Thr465. Multiply glycosylated peptides were observed, as well as variation at individual residues in the nature of the glycan and its presence or absence. Such extreme heterogeneity in the pattern of glycosylation has not been reported previously, and suggests a novel dimension in molecular variation within a bacterial population that may be significant in persistence of the organism in its natural environment. These results demonstrate the usefulness of differential ion mobility in proteomics investigations of PTMs.Glycosylation of flagellin is essential for the virulence of  AGCautomatic gain controlFAIMSfield asymmetric waveform ion mobility spectrometry1Campylobacter jejuni is the most prevalent foodborne bacterial agent of diarrhoeal disease in humans worldwide Virulence factors are not fully understood but it is clear that flagellar motility is crucial in the colonisation by the organism of digestive tracts in birds r determined by SDS‐PAGE is about 10% higher, due to the presence of numerous glycan residues: for an overview see Flagellins FlaA  and FlaB, the major structural protein monomers of the flagellar filament, are proteins each of 572 amino acid residues totalling approximately 59 kDa, although apparent MCampylobacter jejuni  and Campylobacter coli (VC167 T2) by Thibault et al. C. coli (VC167) revealed the presence of legionaminic acid and derivatives Campylobacter jejuni 11168, revealing that the protein was modified by dimethyl glyceric acid derivatives of pseudaminic acid and acetamidino pseudaminic acid at Ser181, Ser207 and either Thr 464 or Thr465. That work was in agreement with the work of Logan et al. who first identified these novel glycans C. jejuni 11168, there was an extensive region of the protein (residues 387–463) for which no coverage was achieved. This observation is particularly salient as the homologous region in flagellin from C. jejuni 81–176 is heavily glycosylated MS was first applied to the analysis of glycosylation of flagellins from Campylobacter jejuni 11168 by use of this multienzyme differential ion mobility approach.In order to address the limited flagellin coverage obtained with trypsin, we have applied an alternative protease for the analysis of flagellin, that is, proteinase K. Proteinase K has a broad specificity and cleaves peptide bonds that are C‐terminal to aliphatic and aromatic residues 22.1Campylobacter jejuni (NCTC 11168) cultures were grown on Mueller Hinton agar  and incubated in microaerophilic conditions  for 30 h. Cells were harvested from 13 separate agar plates and suspended in 5.7 mL of Luria‐Bertani Broth. The suspension was homogenised at high speed (20 500 rpm) using an Ultra Turrax T‐25 homogeniser, for 2 min, with 30 s rest, for four cycles in order to shear the flagellar filament from the bacterial wall. The homogenate was centrifuged at 7500 rpm for 20 min at 4°C to remove cells and cell debris. The supernatant was collected and treated with 1% Triton X (Bio‐Rad), incubated at 37°C for 45 min, and subjected to ultracentrifugation  at 50 000 rpm for 1 h at 4°C. The supernatant was removed immediately, tubes inverted on filter paper to remove residual fluid, and the resulting pellets were re‐suspended in 100 μL distilled water. A 5 μL aliquot of the sample was analysed by SDS‐PAGE, and visualised using Coomassie stain R250 (Bio‐Rad) to check flagellar protein purity, see Supporting Information Fig. 1. A further 5 μL was subjected to a Bradford protein assay to measure the protein concentration. The remaining sample was stored at –20°C until further analysis. Two biological replicates were considered.2.2For LC MS/MS analysis, 10 μL of sample (∼7 μg of the extracted flagellar protein) was made up to 100 μL in 100 mM of ammonium bicarbonate ) and denatured by incubating at 95°C for 5 min. Trypsin  ) was added to the mixture to give 1:50 (replicate#1) or 1:7 (replicate#2) ratio of enzyme to protein, and incubated overnight at 37°C with mild shaking. Proteolysis was quenched by freezing at –20°C and the sample stored until further analysis. For LC FAIMS MS/MS analysis, 50 μg of protein were digested using the digestion procedure as above.2.32 ) was added to the mixture to give a 1:1 (replicate#1) or 1:100 (replicate#2) ratio of enzyme to protein, and incubated for 1 h (replicate#1) or 2 h (replicate#2) at 37°C with mild shaking. Proteolysis was quenched by freezing at –20°C and the sample stored until further analysis.Approximately 84 μL of the sample (70 μg of the extracted flagellar protein) was made up to 100 μL in 100 mM of ammonium bicarbonate  and denatured at 95°C for 5 min. Proteinase K  , 5 mM CaCl18 column zip tips  (according to manufacturers’ instructions). Briefly, the tips were wetted using 100% acetonitrile , and then equilibrated using 0.1% trifluoroacetic acid . The samples were aspirated and dispensed for 10 cycles, and then washed three times using 0.1% TFA and eluted using 70:30 acetonitrile and 0.1% TFA. The desalted samples were dried and re‐suspended in 10 μL of 0.1% formic acid prior to MS analysis.Both trypsin‐ and proteinase K‐treated samples were desalted using C2.418 column and separated by a 30 min gradient from 3.2 to 44% of mobile phase B, followed by 10 min at 90% of mobile phase B and 16 min with 3.2% mobile phase B to re‐equilibrate the system. The LC was coupled to an Advion Triversa Nanomate  that infused the peptides at a spray voltage of 1.7 kV. Peptides were infused directly into the Thermo Orbitrap Velos ETD mass spectrometer (Thermo Fisher Scientific) at a flow rate of 0.35 μL/min.Peptides were separated using online reversed phase LC (Dionex Ultimate 3000) using a binary solvent system consisting of mobile phase A /0.1% formic acid ) and mobile phase B /0.1% formic acid ). Six microliters of the desalted samples were loaded onto a 150 mm Acclaim® Pepmap 100 Cm/z 380–1600) and subsequent CID and ETD of the four most abundant precursor ions. Survey scans were acquired in the Orbitrap with a resolution of 60 000 at m/z 400. Only multiply charged precursor ions were selected for MS/MS. The dynamic exclusion was used with a repeat count of 1 for 30 s. Automatic gain control (AGC) was used to accumulate sufficient precursor ions. AGC target value for FT‐MS was 1 × 106 charges. CID was performed in the linear ion trap using helium at normalised collision energy of 35%. Width of the precursor isolation window was 2 Th. AGC target was 5 × 104 charges with maximum injection time of 100 ms. Charge state‐dependant ETD was performed in the linear ion trap with activation time 100 ms. Isolation window was 3 Th. AGC target of ETD was performed with fluoranthene ions. AGC target was 5 × 104 charges with maximum injection time of 100 ms. Supplemental activation was enabled with activation energy of 25%. Data acquisition was controlled by Xcalibur 2.1 (Thermo Fisher Scientific).The mass spectrometer alternated between a full FT‐MS scan (2.5The LC was coupled to the FAIMS device (Thermo Fisher Scientific), though a modified nanospray HESI II heated electrospray source, similar to that used by Swearingen et al. 2.6b and y ions for CID and c, y and z ions for ETD). Data were searched against a manually created Campylobacter flagellin database (886 sequences) or a Campylobacter flagellin 11168 database (59 sequences) with sequences from the NCBI nr database. The data were searched using both the SEQUEST and Mascot algorithms . In both SEQUEST and Mascot searches, the following parameters were used: precursor ion m/z tolerance 10 ppm; fragment ion tolerance 0.5 Da; enzyme—either trypsin or nonspecific enzyme (proteinase K); maximum number of missed cleavages = 2; no fixed modifications; dynamic modifications as described below. Multiple searches of the data were performed with different combinations of dynamic modifications (maximum number of dynamic modifications per search = 6). The glycans considered were as described in 13H20N2O7, + 316.127 Da, (S/T), C16H27N3O8 + 389.180 Da (S/T), C13H21N3O6 + 315.143 Da (S/T) and C16H26N2O9 + 390.164 (S/T); (B) oxidation +15.995 Da (M), C16H27N3O8 + 389.180 Da (S/T), C16H26N2O9 + 390.164 (S/T), C15H24N2O11 + 408.138 Da (S/T), C20H31N5O9, +485.212 Da (S/T), and C15H22N2O8, + 358.137 Da (S/T); (C) oxidation +15.995 Da (M), C16H27N3O8 + 389.180 Da (S/T), C16H26N2O9 + 390.164 (S/T), C13H20N2O7 + 316.127 Da (S/T), C20H30N4O10 + 486.196 Da (S/T) and C14H23N3O6 + 329.1587 (S/T); (D) oxidation +15.995 Da (M), C13H20N2O7 +316.127 Da (S/T), C13H21N3O6 + 315.143 Da (S/T), C15H24N2O11 + 408.138 Da (S/T), C20H31N5O9, +485.212 Da (S/T), and C15H22N2O8, + 358.137 Da (S/T); (E) oxidation +15.995 Da (M), C13H20N2O7 +316.127 Da (S/T), C13H21N3O6 + 315.143 Da (S/T), C20H30N4O10 + 486.196 Da (S/T) and C14H23N3O6 +329.1587 (S/T) and C16H26N2O9 + 390.164 (S/T); (F) oxidation +15.995 Da (M), C16H27N3O8 + 389.180 Da (S/T), C16H26N2O9 + 390.164 (S/T), C13H20N2O7 +316.127 Da (S/T), C13H21N3O6 + 315.143 Da (S/T), and C15H24N2O11 + 408.138 Da (S/T). Glycopeptide identities were confirmed by manual analysis using the MS product package in Protein Prospector (http://prospector.ucsf.edu/prospector/mshome.htm).Raw data were loaded into Proteome Discoverer (version 1.4). Precursors with a mass of less than 350 Da and greater than 5000 Da were excluded and MS/MS spectra were required to have at least 1 peak. The CID and ETD spectra were separated and searched with the same parameters except for the fragment ions ; dimethylglyceric acid derivative of 7‐acetamidino pseudaminic acid , pseudaminic acid , acetamidino pseudaminic acid/legionaminic acid . The structures of these glycans are shown in Scheme In our previous paper The combined protein sequence coverage obtained from the two replicates with CID was 76.2%. Eighteen glycopeptides were identified in the two database searches, however manual analysis of the mass spectra revealed that in each case fragmentation was poor and the peptide sequence coverage insufficient to assign sites of glycosylation. The combined protein sequence coverage obtained for ETD was 76.2%, see Fig. Campylobacter jejuni 11168 by this glycan has not been observed previously.Seven of the glycopeptides identified were modified by Δm389 or by Δm390, five of which were at Ser181, Ser207 and Thr 464 or Thr465. These combinations of glycan and modification site were seen in our previous work. Two of the glycopeptides were glycosylated by these glycans at Ser345 and 349. These sites of glycosylation have not been observed previously. Two of the glycopeptides are modified by Δm315, one at Ser181 and one at Ser207. Modification of flagellin A from C. jejuni 81–168 As observed previously, no sequence coverage was observed between amino acid residues 387 and 463. This region of the protein has a high concentration of serine and threonine residues and may be heavily glycosylated, as observed in 3.2Trypsin digests of flagellin were analysed by use of LC FAIMS MS/MS with the top‐4 MS method. An ‘external CV stepping’ FAIMS method C. jejuni 11186.Seven glycopeptides containing Ser181 were identified, four of which were modified by Δm389 or Δm390, as observed previously Five glycopeptides containing Ser207 were identified, four of which were modified by Δ389 or Δm390. The remaining peptide was modified by Δm315 at Ser 207. This combination of glycan/glycosylation site has not been observed previously. Finally, three peptides were identified which contained Thr464 and Thr465, modified by Δm389, Δm390 and Δm315. In all cases, although the ETD fragmentation was extensive, no cleavage between the two threonine residues was observed precluding unambiguous site localisation. As seen above, glycosylation of these site(s) by Δm389 and Δm315 are novel observations.The introduction of FAIMS into the workflow resulted in observation of novel glycans and combinations of glycan/glycosylation sites. Nevertheless, protein coverage between amino acid residues 388 and 463 was not achieved, presumably as a result of the use of trypsin as the protease.3.3To address the problem of the ‘missing region’, i.e. amino acid residues 388–463, an alternative protease—proteinase K —was considered. Two biological replicates were investigated. For the first, proteolysis conditions were enzyme/protein ratio 1:1, 1 h incubation (replicate #1). Subsequent experiments to determine the optimum conditions (to achieve maximum coverage) for proteolysis by proteinase K were performed (data not shown), and conditions found to be enzyme/protein ratio 1:100, 2 h incubation (replicate #2). The proteinase K digest of flagellin was first analysed by the ‘top‐4’ LC MS/MS method without FAIMS. The combined protein sequence coverage obtained for ETD was 18.4%, see Fig. Twenty glycopeptides were assigned in the database search of the ETD data, however manual analysis of the ETD spectra, with cross‐validation against the corresponding CID spectra, revealed 17 true assignments, see Table 3.4m/z 316 and 390. This peptide was observed at compensation voltages –45 V and –50 V (as 3+ precursor). Differential glycosylation of this peptide was observed, i.e. modification at Ser395 and Ser398 with glycans Δm315 and Δm390, respectively. That peptide was observed at CV = –45 V (3+ precursor) and –25 V (2+ precursor).Proteinase K digests were analysed by LC FAIMS MS/MS  with the top‐4 MS method. The combined protein sequence coverage obtained for ETD was 39.0%, see Fig. m/z 316, 317, 390 and 391 suggesting both species are present; however, the relative abundances of the oxonium ions suggests that (ii) is the predominant species. It is not possible to confirm unambiguously the sites of the Δm315 and Δm316 glycans within the glycopeptide because of the potential for hydrogen transfer between ETD fragments. The glycans are sited on Ser448 and Ser454 (or a combination of the two). Figure The ETD spectrum shown in Fig. Figure 4Campylobacter jejuni 11168 may be achieved by incorporating differential ion mobility spectrometry into the bottom‐up proteomics workflow, together with use of both trypsin and proteinase K for proteolysis. A summary of the sites of glycosylation observed is given in Fig. We have demonstrated that comprehensive mapping of the O‐glycosylation of flagellin from The authors have declared no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Figure S1. SDS‐PAGE analysis of purified Campylobacter jejuni flagellin protein. 10% SDS‐PAGE gel, stained with Coomassie blue. Lane 1 – MW markers. Lane 2 – cell suspension from C. jejuni strain 11168 culture, Lane 3 – purified flagellin proteinFigure S2.Figure S3.Figure S4.Figure S5.Supplemental Table 1: Non‐glycopeptides identified from tryptic digest of flagellin following ETD MS/MS (with and without FAIMS). .Supplemental Table 2: Non‐glycopeptides identified from proteinase K digest of flagellin following ETD MS/MS (without FAIMS). .Supplemental Table 3: Non‐glycopeptides identified from proteinase K digest of flagellin following ETD MS/MS (with FAIMS). .Comprehensive mapping of O‐glycosylation in flagellin from Campylobacter jejuni 11168: A multi‐enzyme differential ion mobility mass spectrometry approachClick here for additional data file.


The chains characterizing the alluaudite structure are built up from edge-sharing  octahedra stacked parallel to [10-1]. These chains are held together by VO4 tetrahedral groups, forming polyhedral sheets perpendicular to [010]. Within this framework, two types of channels extending along [001] are present. One is occupied by (Na+/Mn2+) while the second is partially occupied by Na+. The mixed site containing (Na+/Mn2+) has an octahedral coordination sphere, while the Na+ cations in the second channel are coordinated by eight O atoms.The title compound, sodium  triiron tris[vanadate(V)], (Na A(1)A(2)M(1)M(2)2(XO4)3. The M(1) and M(2) sites accommodate di- or trivalent cations in an octahedral environment and are connected to the tetrahedral XO4 groups, leading to an open-framework structure. Alluaudite-type phosphates are of special interest as positive electrode materials in lithium and sodium batteries. For instance, the alluaudite-type lithium manganese phosphate Li0.78Na0.22MnPO4 is proposed by Kim et al. (20142Fex3-Mnx(PO4)3, the electrochemical performance is associated either with morphology or with the electronic and crystalline structure 2PO4 2PO4 3 3  with approximate molar ratios of A:M:M′:V = 2:2:1:3 and report here details of the preparation and structural characterization of the first sodium- manganese- and iron-based vanadate with an alluaudite-type structure, viz. (Na0.70)2Fe2+(VO4)3.Besides well-known phosphate phases, arsenates ; Na1+ and Mn12+ are disordered in a 0.7:0.3 ratio on a site located on an inversion centre (4b), and Na2+ is present at a site on a twofold rotation axis (4e) with 70% occupancy. All other sites are fully occupied. Nearly the same cationic distribution was reported by Yakubovich et al. 2Fe2+(PO4)3.The preparation of this compound by melting a mixture of three metal oxide precursors in addition to vanadium oxide forced us to explore several crystallographic models. Refinement of the occupancy ratios, bond-valence analysis and the electrical neutrality requirement of the structure lead to the given composition for the title compound. The basic building units of the structure are shown in Fig. 1 al. 1977 for the 6] octahedra, leading to the formation of kinked chains running along   and statistically occupied Na2+ cations are accommodated  site has a distorted octahedral oxygen environment, with —O bond lengths between 2.4181 (16) and 2.5115 (15) Å. The Na2+ cation is coordinated by eight oxygen atoms with Na2—O distances in the range 2.4879 (18) to 2.982 (3) Å. The disorder of Na+ in the channels might admit ionic mobility for this material.The crystal structure of the title compound is built up from edge-sharing [FeO0] Fig. 2. These c0] Fig. 3. Therebyed Fig. 4. The  site, full occupation was assumed, with the sum of the site occupation factors constrained to be 1. The site-occupation factor of Na2+ was refined freely. In the last step of the refinement, the site occupation factors were fixed to fulfill electro-neutrality. Reflection (1 5 0) was probably affected by the beam-stop and was omitted from the refinement. The remaining maximum and minimum electron density peaks are located 0.59 and 0.41 Å from Fe2 and V2, respectively.Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2056989016000931/wm5259sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016000931/wm5259Isup2.hklStructure factors: contains datablock(s) I. DOI: 1447912CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The electronic structures of mononuclear and dinuclear iron(iv) complexes are studied using magnetic circular dichroism and wavefunction-based ab initio methods, and then correlated with their similar reactivities toward H- and O-atom transfer. iv)-oxo species are key intermediates in the catalytic cycles of a range of O2-activating iron enzymes. This work presents a detailed study of the electronic structures of mononuclear ([FeIV(O)(L)(NCMe)]2+, 1, L = trisamine) and dinuclear ([(L)FeIV(O)(μ-O)FeIV(OH)(L)]3+, 2) iron(iv) complexes using absorption (ABS), magnetic circular dichroism (MCD) spectroscopy and wave-function-based quantum chemical calculations. For complex 1, the experimental MCD spectra at 2–10 K are dominated by a broad positive band between 12 000 and 18 000 cm–1. As the temperature increases up to ∼20 K, this feature is gradually replaced by a derivative-shaped signal. The computed MCD spectra are in excellent agreement with experiment, which reproduce not only the excitation energies and the MCD signs of key transitions but also their temperature-dependent intensity variations. To further corroborate the assignments suggested by the calculations, the individual MCD sign for each transition is independently determined from the corresponding electron donating and accepting orbitals. Thus, unambiguous assignments can be made for the observed transitions in 1. The ABS/MCD data of complex 2 exhibit ten features that are assigned as ligand-field transitions or oxo- or hydroxo-to-metal charge transfer bands, based on MCD/ABS intensity ratios, calculated excitation energies, polarizations, and MCD signs. In comparison with complex 1, the electronic structure of the FeIV PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="34" y-size="34" xml:space="preserve"> 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000	</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="16.000000pt" height="16.000000pt" viewBox="0 0 16.000000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O site is not significantly perturbed by the binding to another iron(iv) center. This may explain the experimental finding that complexes 1 and 2 have similar reactivities toward C–H bond activation and O-atom transfer.High-valent iron( For clarity, hereafter we refer to the FeIVO site of complex 2 as FeA, and the FeIV–OH site as FeB.Over fifty mononuclear iron and excited state spectroscopy (electronic absorption spectroscopy (ABS) and resonance Raman spectroscopy).ε) between the left (LCP) and right (RCP) circular polarized light of a sample is measured in the presence of a longitudinal magnetic field (relative to the light beam). MCD intensity arises from three different mechanisms designated as A-, B- and C-terms,10 and the following relationship is observed:10In MCD spectroscopy, the difference in the absorption  and levels off to its saturation limit. For S = 1/2 systems with orbitally non-degenerate ground states, the magnetization behavior follows a hyperbolic tangent and magnetization curves at all temperatures overlay. In the case of S > 1/2 systems, the magnetization curves do not superimpose and show a complicated nested behavior. The general theory to calculate the signs and intensities of MCD C-term transitions for S ≥ 1/2 has been simultaneously and independently worked out by Oganesyan et al.11 and Neese and Solomon.12 Based on this formalism, one can determine ground-state spin-Hamiltonian (SH) parameters and the polarizations of the respective electronic transitions through fitting the nested magnetization curves obtained from variable-temperature variable-field (VTVH) MCD experiments. This method has been successfully applied to analyze MCD spectra of mono-13 as well as di-nuclear transition metal complexes.14As the temperature decreases and/or the magnetic field increases, it is frequently observed that the MCD C-term intensity can be simulated using the following equation,12 is the MCD intensity, γ is a collection of constants, S is the total spin of the ground state, Ni is the Boltzmann population of the ith magnetic sublevel of the electronic ground state, lx,y,z are the directional cosine values of the angles between the magnetic field and the molecular coordinate system, ŝx,y,zi are the expectation values of the x,y,z component of the spin operator ŝ over the ith magnetic eigenstate, respectively. The Meffvw's  are the effective transition dipole moment products and are given by12LMNt is related to the reduced matrix element of spin–orbit coupling (SOC) operator, ΔMN is the energy separation of state M relative to N, and DMNt is the electric transition dipole moment along t-direction. Moreover, the x, y and z fractional polarization factors can be computed as follows,y and z polarized fractions of the intensity.It has been shown that for paramagnetic systems VTVH MCD g, D and E enter the model through the spin expectation values and the Boltzmann populations of the various magnetic sublevels, and can therefore be calculated.The SH parameters 15 This lies in the fact that MCD spectra typically exhibit higher resolution than corresponding ABS spectra especially in the presence of multiple overlapping absorption bands, because MCD features are signed quantities. In order to fully exploit the information content present in the MCD spectrum, one can determine the individual MCD signs of electronic transitions of interest. This will definitely lower the possibility of erroneous band assignments and provide more insight into electronic structures. However, only a few examples concerning the determination of MCD signs have been reported so far,16 due to the stricter selection rules for MCD compared to ABS spectroscopy. It has been shown that for a system with orbitally non-degenerate ground and excited states, the MCD C-term intensity arises from SOC between the excited states J and K (J–K coupling) and between ground state A and excited state K (A–K coupling) , because the two MCD signals in principle have opposite signs and similar excitation energies. The absolute signs of the A → J and A → K transitions depend on the symmetry of the states A, J, K. If the transition is dominated by a single excitation, the MCD signs can be determined by the symmetry of the electron donating orbital (EDO) and the electron accepting orbital (EAO).It is by no means straightforward to interpret MCD spectra and extract electronic structure information about excited states. Therefore, in most cases, MCD spectroscopy is employed as a complementary technique to conventional ABS spectroscopy for resolving and assigning electronic transitions.oupling) .11a,12A-, B-, and C-terms by Stephens et al., perturbational calculations of these terms were attempted. Recently, Seth, Ziegler and co-workers reported an elegant formulation of MCD C-term intensity based on linear response perturbation expressions of three MCD terms (eqn (1)) using time-dependent density functional theory.17 This work presents definite progress in the computational approach to MCD. However, it does not solve the fundamental problem that with DFT approaches one is unable to represent the all-important magnetic sublevels Ms = S, S – 1, …, –S of a system with a total spin S explicitly. Furthermore, for a high-spin system (S > 1/2) the very limited accuracy of DFT in the prediction of zero-field splitting (ZFS) parameters will affect MCD calculations. In addition, these approaches are only applicable in the linear response regime with respect to the magnetic field strength as implied by eqn (1). More importantly, single determinant methods such as density functional theory fail in the description of multiplet effects or double excitations, both of which are prevalent in the optical spectra of transition metal complexes. In this regard, one has to resort to multi-reference approaches18 such as the complete active space self-consistent field (CASSCF)19 and second-order N-electron valence perturbation theory (NEVPT2).20 Our approach to calculate MCD signs and intensities is to directly estimate the difference between LCP and RCP transition probabilities in the presence of a homogeneous external magnetic field using exact diagonalization rather than perturbation theory. Specifically, transition energies and intensities for LCP and RCP light are computed using ground and excited state wavefunctions obtained from quasi-degenerate perturbation theory (QDPT) treatment, which explicitly accounts for SOC, spin–spin coupling (SSC), and Zeeman interactions.21 The method was tested successfully for diatomic molecules as well as for simple transition metal complexes.21 Here we report the first application of this methodology to more complex mono- and dinuclear transition metal compounds.The best way to quantitatively interpret experimental MCD spectra is to compute them using quantum chemical approaches. The theoretical prediction of MCD spectra has been proven to be rather challenging. Based on the formulation of 1 and 2 were prepared with 1 : 4 acetonitrile–butyronitrile (MeCN–PrCN). Upon freezing, this solvent mixture becomes a transparent glass, making it suitable for MCD measurement. Anhydrous MeCN were purchased from Aldrich and used as received. PrCN (99%+) purchased from Aldrich was purified and dried according to reported procedures.22 2.2 mM [FeII(L)(MeCN)2](OTf)2 in 1 : 4 MeCN–PrCN was maintained at –60 °C and treated with 2 equivalents of 2-(tert-butylsulfonyl)-iodosylbenzene, similar to a reported procedure for preparation of oxoiron(iv) complexes.23 Complex 1 formed in 2 minutes with ∼90% yield, as indicated by its absorption at 720 nm.24 4.4 mM solution of 2 in MeCN was prepared according to a reported procedure,9 then diluted with 4 equal volumes of cold PrCN. The solutions were transferred to pre-cooled MCD holders with pre-cooled syringes, and then frozen in liquid nitrogen.MCD samples of complexes –1 (2000 nm) to 30 000 cm–1 (333 nm). At the peak positions we carried out VTVH MCD experiments in which the MCD intensity is measured at the fixed wavelength as a function of temperature and magnetic field. The results are presented as plot of MCD intensity vs. βB/kT, where β is the Bohr magneton, k is Boltzmann's constant, B is strength of the applied magnetic field and T is absolute temperature.MCD experiments were carried out with an Olis DSM17 CD spectrapolarimeter while the sample was placed in the Oxford cryostat Spectromag SM4000. The temperature range was from 2 K to 60 K and the energy range from 5000 cm25 Given the size of the complexes under investigation, the substituents on the pyridine ring were replaced by hydrogen atoms, viz. an unsubstituted TPA ligand (TPA = tris(2-pyridyl-methyl)amine) was employed in the calculations. Geometry optimizations were carried out using the hybrid B3LYP density functional26 along with the semi-empirical van der Waals corrections due to Grimme.27 Def2-TZVP(–f)28 basis sets on Fe, O, and N, and def2-SV(P)29 on the remaining atoms were used. The density fitting and “chain of spheres” (RIJCOSX)30 approximations were employed to accelerate the calculations in conjunction with the auxiliary basis sets def2-TZV/J.31 Solvation effects were taken into account by using the conductor like screening model (COSMO).32 Consistent with experiment, acetonitrile was selected as the solvent in the calculations.All calculations were performed with the ORCA program package.1. In the CASSCF calculations, an active space consisting of twelve electrons in the five Fe-3d based molecular orbitals (MOs), the three oxo-p orbitals and the bonding counterpart of the Fe-dx2–y2 orbital ) was chosen. However, the CASSCF results erroneously predict a quintet ground state, because CASSCF is designed to mainly capture static correlation energies and hence lacks balanced treatments between static and dynamic correlation effects. Using NEVPT2, dynamic correlation effects were explicitly introduced. It turns out that the NEVPT2 calculations deliver the correct spin-state energetics for complex 1; therefore, in the following we will only discuss the NEVPT2 results.The CASSCF/NEVPT2 approach was used to compute the MCD spectra of complexes IV site of complex 2 were employed, the active space for the entire complex ) would be too large to render usual CASSCF calculations feasible. Using density matrix renormalization group (DMRG)33 approach, exploring CASSCF ) calculations on a model complex in which TPA is replaced with four NH3 ligands suffered from severe convergence problems. More importantly, current DMRG calculations only deliver excitation energies and are not able to provide the information about the nature of excited states and hence the assignment of transitions. Given these issues, complete active space configuration interaction (CASCI) is the only method of choice, since time-dependent DFT (TD-DFT) yields qualitatively unreasonable results (vide infra). To circumvent the problem of the size of active spaces, we carried out CASCI calculations with a series of active spaces. CAS is composed of the five d-orbitals in each iron center and the two p-orbitals of the bridging oxo ligand (OB) that form π-bonds with the t2g-derived d-orbitals of FeB, and CAS consists of the five d-orbitals in each iron center and the three p-orbitals of the terminal oxo group (OT).If a similar active space ) for each Fe1 exhibits a broad feature centered around 13 800 cm–1 in the near-IR region . At higher temperatures, the intensities of the three bands decrease due to the usual MCD C-term behavior with increasing temperatures (TMC)(NCMe)]2+, [FeIV(O)(TMC)(OC(O)CF3)]2+ and [FeIV(O)(N4Py)]2+ -methyl)-N,N-bis-(2-pyridylmethyl)-amine).34 In addition, a broad band centered around 18 200 cm–1 (band 4) and another intense negative band at 24 200 cm–1 (band 5) are found for complex 1. Our variable temperature (VT)   and S2† 1 is situated in a distorted octahedral environment with a characteristic Fe–oxo bond distance of 1.67 Å.8 Similar to other well-characterized ferryl species,7 this short metal–ligand bond distance reflects the rather covalent nature of the Fe–oxo interaction, which consists of two half π-bonds  and one σ-bond .35 The orbital occupation pattern of the triplet ground state for complex 1 can be described as σ(O-pz)2π4δ(Fe-dxy)2π*2σ*(Fe-dx2–y2)0σ*(Fe-dz2)0 (2(Fe-dxy) to nearly degenerate singly occupied molecular orbitals (SOMOs) 2e leads to a 3E(1b2 → 2e) excited state. Due to multiplet effects, the excitations from 1b2(Fe-dxy) to the virtual molecular orbitals (VMOs) 2b1(Fe-dx2–y2) and 2a1(Fe-dz2) result in a series of excited states as shown in 36 Specifically, if the two electrons in the 2e-set have parallel spin, the excited states are of 3A1(1b2 → 2b1) (× 2), and 3B1(1b2 → 2a1) (× 2) symmetry. On the other hand, if the two electrons in the 2e-set have opposite spin, the excited states of 3A2(1b2 → 2b1), 3B1(1b2 → 2b1), 3B2(1b2 → 2b1), and 3A1(1b2 → 2a1), 3A2(1b2 → 2a1), 3B1(1b2 → 2a1) symmetry, respectively, arise. Promotion of one electron from the 2e-set into the 2b1(Fe-dx2–y2) and 2a1(Fe-dz2) orbitals gives rise to 3E(2e → 2b1) and 3E(2e → 2a1) excited states.As analyzed in an earlier study, the iron center in complex dz2)0 . The keyIV(O)(NH3)4(H2O)]2+ only deliver two roots for the excitations of (1b2 → 2b1) and (1b2 → 2a1), instead of five as required by spin-coupling algebra.36 To circumvent this problem, explicitly spin-coupled multi-reference approaches such as CASSCF and NEVPT2 have to be employed. Our NEVPT2 calculations not only yield quite accurate ligand field transition energies with a deviation of ∼3000 cm–1 relative to experiment (vide infra).TD-DFT is a method based on the linear response formalism starting from the single Kohn–Sham determinant of the ground state, and is hence unable to properly describe multiplet effects and spin-coupling. As a consequence, it has been reported that the TD-DFT calculations on 2+ (TMG3tren = N[CH2CH2NC(NMe2)2]) complex, and the sign of this pseudo-A term signal is also negative.16 Due to the low intensity of this pseudo-A term signal, the adjacent intense CT transitions may substantially distort its band shape via out-of-state SOC and/or directly obscure it. Our theoretical results suggest that the onset of a series of the ligand-to-metal charge transfer (LMCT) transitions from the oxo group is around 22 430 cm–1; however, the dipole-allowed LMCT transition appears at ∼30 000 cm–1, which is significantly higher in energy than that of band 5. This is due to the lack of proper treatment of dynamic correlation, especially the radial correlation resulting from the change in the metal oxidation state in CT processes.37 To account for this effect, the active space has to be enlarged to include the metal double d-shell.38 Moreover, the intensity variation with different temperatures indicates band 5 to be largely x,y-polarized  corresponds to the feature around 7200 cm–1 in the near infrared (NIR) MCD spectra . At least ten Gaussian transitions are necessary in order to achieve a satisfactory fit of the MCD and absorption spectra. The fitting parameters are collected in C0/D0 values (>0.17) indicate bands 2–5, 7 and 8 to be d–d transitions in nature, whereas the remaining bands  are assigned to CT transitions.In contrast to complex spectrum , top. Th spectra , bottom. spectra  using th2 are presented in  for a ferromagnetically exchange-coupled dimer of two local spins Si = 1 with ZFS and exchange parameters as discussed below A to those found previously for [FeIV(O)(TPA)(NCMe)]2+ .8 Even with these constraints, our simulations reveal that the error surface of the exchange constant J is rather flat; thus, it is not possible to obtain the J value with high precision. In fact, the choice of a J value in the range of 20–80 cm–1 does not discernibly deteriorate the quality of the fits. In this range of the J value, the fitted D parameter of the FeB site changes from 5.3 to 17.2 cm–1, while E/D is always close to 1/3. A satisfactory simulation is presented in gx,y,z = 2.00, DB = +5.3 cm–1 (E/D)B = 0.30 and J = 35 cm–1. The obtained SH parameters are in reasonable agreement with those determined independently by Mössbauer spectroscopy diiron(iv) core where one of the two iron(iv) centers is bound to a terminal oxo ligand (FeAO) and the other is coordinated to a terminal hydroxo group (FeB–OH); FeB–OH forms a hydrogen bond with FeAO  very well. As analyzed previously,42 complex 2 features a quintet ground state arising from ferromagnetic coupling between the two S = 1 iron(iv) sites. To be consistent, we chose the same reference frame, the principal axes of the FeA ZFS tensor, to label the d-orbitals of the two iron sites. Since the angle of FeA–OB–FeB is ∼130°, Z is roughly parallel to FeB–OH. The geometric structure of the FeA site closely resembles that of complex 1 except that the equatorial MeCN is replaced by the bridging oxo group. In fact, the computed FeA–OB distance (1.931 Å) is close to the Fe–NMeCN distance (1.982 Å) calculated for complex 1, indicating much weaker bonding between FeA and OB than one expected. For comparison, Collins and co-workers reported Fe–Ob distances of 1.73–1.74 Å for the only crystallographically characterized (μ-oxo)diiron(iv) complex to date.44 In line with this, the FeA-dxy based MO is a weakly π-antibonding orbital and is hence doubly occupied. Therefore, the electron configuration of FeA site is δ(FeA-dxy)2π*2σ*(FeA-dx2–y2)0σ*(FeA-dz2)0 . This bond length is also shorter than that of FeB–OOH (1.814 Å), reflecting the difference in Lewis basicity between oxo and hydroxo groups. Thus, the FeB-dxy-based MO lies highest in energy among the three FeBt2g-derived MOs and is thus singly occupied. Our calculations show that there exist two alternative electron configurations for complex 2 depending on the relative energies of the two remaining FeBt2g-derived MOs:42 (a) π*(FeB-dxz)2π*(FeB-dyz)1π*(FeB-dxy)1σ*(FeB-dx2–y2)0σ*(FeB-dz2)0 (shown in B-dyz)2π*(FeB-dxz)1π*(FeB-dxy)1σ*(FeB-dx2–y2)0σ*(FeB-dz2)0 with the former configuration lying ∼2 kcal mol–1 lower in energy. For configuration a), because the FeB–OOH–H angle (106°) is close to a right angle, the OOH-px orbital cannot interact strongly with the FeB-dxz orbital but instead predominantly interacts with the H-s fragment orbital. As a result, the FeB-dxz based MO becomes stabilized to some extent relative to the FeB-dyz centered MO. In configuration (b) the FeB–OOH–H angle is ∼140°, this lowers the energy of the FeB-dyz based MO.Complex th FeAO . In the dz2)0 , which ishown in  and b) π2  along with significant rhombicity (E/D > 0.3).42 The smaller C0/D0 ratio of band 6 leads us to assign this band as a CT transition. Our calculations suggest that it arises either from the bridging oxo group to the FeB site or from the terminal OH group to FeB. The latter assignment agrees with the polarization of band 6 deduced from the VTVH analysis. Based on the computed excitation energies, we tentatively assign bands 7 and 8 as the t2g → dz2 transitions of FeA and FeB sites. Given the C0/D0 ratio values, bands 9 and 10 are certainly CT transitions from the terminal oxo and hydroxo groups and/or the π-systems of TPA ligand to both iron centers. Our computations predict the wrong signs of bands 9 and 10. This may be attributed to the unbalanced treatment of the dynamic and static correlation in CT processes, similar to the situation encountered for band 5 of complex 1.The calculated MCD spectrum of complex  Fig. S12 is overaig. S122 , we can  Fig. S12. Bands 3iv)-oxo complexes, which involves the transfer of an electron from the σC–H bond that is cleaved to the dxz/yz- (π-pathway) or dz2-based MOs (σ-pathway) of the ferryl center and proton transfer to the oxo group  site does not discernibly change the local electronic structure of the reactive iron(iv)-oxo unit in complex 2, consistent with the weak exchange interaction between the two Fe(iv) centers in complex 2. Moreover, the local coordination environments of the Fe(iv)O moiety in both complexes are similar, as they are supported by the same auxiliary ligand and the interaction between FeA and the bridging oxo ligand is relative weak (vide supra). Thus, one can anticipate that both complexes may suffer from similar energetic penalties arising from the electronic structure rearrangement and the resulting geometric distortions in the HAT process. As such, subtle effects such as hydrogen bonding may have a significant influence on the relative reactivities of the two complexes. Our previous work showed that the one-electron reduced form of complex 2 ([(L)FeIV(O)(μ-O)FeIII(OH)(L)]3+) is 10-fold less reactive toward H-atom transfer than [(L)FeIV(O)(μ-O)FeIII(F)(L)]3+, both of which share a similar core structure and feature the same electronic structure.46 The differential reactivity was attributed to the presence of a hydrogen bond between the terminal hydroxo and oxo groups in the former complex similar to that found for complex 2, which entails an additional activation barrier for breaking this hydrogen bond during the reaction. Correspondingly, the experimental finding that the HAT reaction rate by complex 1 is five times faster than that by complex 2 can be readily rationalized.9H-atom transfer (HAT) is found to be the rate-determining step for C–H bond oxidation by iron-oxo species, whereas in the case of HAT, both the electron affinity of the oxidant and the proton affinity of its one-electron reduced form govern the reaction rate. Complex 2 has an overall +3 charge versus a +2 charge for 1. The greater electron affinity of 2 due to its increased charge may then compensate for the added barrier required to break the hydrogen bond, to afford an OAT rate comparable to that of 1. On the other hand, for an HAT reaction, the increased electron affinity of an oxidant also leads to a decreased proton affinity,48 so the two factors cancel each other out and the effect of the hydrogen bond is observed.Interestingly, the O-atom transfer (OAT) reactivities do not follow the trend of HAT, but were experimentally found to be the same between the two complexes.1) and a di-nuclear (2) iron(iv) complex supported by the same tetradentate tripodal ligand using a combined experimental and theoretical approach is presented herein. The salient features of the current work are the direct calculation of MCD spectra with wave-function-based multi-reference methods and independent determination of the MCD signs based on the associated EDO and EAO for a given transition. In comparison with experiment, this approach allows us to make unambiguous assignments of the important transitions of complex 1 and gain more insight into the MCD signs and the temperature-dependent intensity variations, both of which aid in our interpretation of the more complicated MCD spectra of complex 2. Based on MCD/ABS intensity ratios, calculated excitation energies, polarizations, and MCD signs, the key transitions of complex 2 are assigned as ligand-field- or oxo- or hydroxo-to-metal charge transfer transitions. The correlation of the electronic structures of complexes 1 and 2 with their reactivities toward C–H bond oxidation and O-atom transfer reveals that, despite a difference in nuclearity, the two ferryl sites actually have very similar electronic structures that lead to similar reactivities.A detailed investigation of the electronic structures of a mono- (


Planktothrix strain. We assessed potential molecular effects of aeruginosin 828A in comparison to another cyanobacterial serine protease inhibitor, cyanopeptolin 1020, in human hepatoma cell line Huh7, in zebrafish embryos and liver organ cultures. Aeruginosin 828A and cyanopeptolin 1020 promoted anti-inflammatory activity, as indicated by transcriptional down-regulation of interleukin 8 and tumor necrosis factor α in stimulated cells at concentrations of 50 and 100 µmol·L−1 aeruginosin 828A, and 100 µmol·L−1 cyanopeptolin 1020. Aeruginosin 828A induced the expression of CYP1A in Huh7 cells but did not affect enzyme activity. Furthermore, hatched zebrafish embryos and zebrafish liver organ cultures were exposed to aeruginosin 828A. The transcriptional responses were compared to those of cyanopeptolin 1020 and microcystin-LR. Aeruginosin 828A had only minimal effects on endoplasmic reticulum stress. In comparison to cyanopeptolin 1020 our data indicate that transcriptional effects of aeruginosin 828A in zebrafish are very minor. The data further demonstrate that pathways that are influenced by microcystin-LR are not affected by aeruginosin 828A.Intensive growth of cyanobacteria in freshwater promoted by eutrophication can lead to release of toxic secondary metabolites that may harm aquatic organisms and humans. The serine protease inhibitor aeruginosin 828A was isolated from a microcystin-deficient  Cyanobacteria  have inhabited our planet for over 3.5 billion years and produce oxygen by photosynthesis. Compared to green algae, cyanobacteria possess distinct advantages, such as fixation of nitrogen and gas vesicles which allow vertical movements. They produce numerous secondary metabolites, which have many different functions including growth inhibition of competing phytoplankton species , increasPlanktothrix rubescens strain 91/1, can successfully defend against grazers. Recently, a sulfate and chlorine containing linear molecule of the aeruginosin family, aeruginosin 828A (AG 828A), was isolated and demonstrated to be toxic to Thamnocephalus platyurus with an LC50 of 22.4 µmol·L−1 pyrene (BaP) in concentrations of 0.1 and 1 µmol·L−1, led to up-regulation, no increase in EROD activity was observed after exposure of Huh7 cells for 24 h to concentrations of 20, 50, and 100 µmol·L−1 AG 828A , CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), spliced X-box binding protein 1 (XBP-1s), growth arrest and DNA damage-inducible protein 34 (GADD34), death receptor 5 (DR5), jun proto-oncogene (CJUN), fos proto-oncogene (CFOS), and caspase 8 (CASP8). The data in BIP, only. For all other genes belonging to different pathways , and lasted until seven dpf. The abundance of transcripts of selected genes related to circadian rhythm, such as nuclear receptor subfamily 1, group D, member 1 (nr1d1) and period 1 (per1), or DNA damage repair, cryptochrome 5 (cry5), did not change (for prostaglandin D2 synthase (ptgds) or the efflux transporter adenosine triphosphate-(ATP) binding cassette transporter sub-family G member 2 (abcg2).Exposure of zebrafish embryos to 0.1 and 1 µmol·Lt change . FurtherCYP1A induction.To evaluate additional toxicological pathways that may be affected by AG 828A, we analyzed the transcription of genes involved in cellular stress responses, detoxification, and MAPK signaling, as well as AHR regulated genes after exposure of zebrafish eleuthero-embryos to AG 828A. Furthermore, alteration of these gene transcripts was assessed in zebrafish liver organ cultures. However, no significant alterations of gene expression were found . These dPlanktothrix strain, it was suggested that this toxin may compensate for the lack of toxic MCs pyrene (BaP)  was used in concentrations of 0.1 and 1 µmol·L−1, as well as the CYP1A and ER stress inducer tunicamycin  at 12 µmol·L−1 [−1 AG 828A and 1 µmol·L−1 BaP in HBSS , or 1 µmol·L−1 BaP  in medium with FBS . Solvent controls of 1% DMSO  were also included.EROD assay was performed to measure cytochrome P450 1A (CYP1A) activity. This assay is based on the conversion of 7-ethoxyresorufin to the fluorescent resorufin by ethoxyresorufin-O-deethylase. The amount of product and therefore activity of CYP1A is determined by fluorescence measurements. Cells were seeded on 96 well plates in culture medium . A 24 h exposure was performed in a first setting at 0, 20, 50, and 100 µmol·Lµmol·L−1 . In a se−1 of the CYP1A substrate 7-ethoxyresorufin  and plates were incubated for 20 min at culture conditions. A Synergy 2 plate reader  was used to measure the fluorescence of the product resorufin using Gen5 software . The exciatation wavelength was 544 nm, the detected emission wavelength 590 nm. After 24 h, the exposure media were replaced by HBSS  containing 1 µmol·L−1. DMSO  was used as solvent with a final concentration of 0.01% in every concentration group. Each group consisted of 4 replicates with 15 eleuthero-embryos each. Eleuthero-embryos were statically exposed until 168 hpf and fixed in RNA Later  afterwards. RNA was extracted, cDNA synthesized, and RT-qPCR was performed (see Section 5.9). The same primers were used as in [At 72 h post fertilization (hpf), hatched zebrafish embryos were transferred to 48 well plates (one eleuthero-embryo per well)  containing reconstituted fish water. The water was subsequently replaced by 450 µL of exposure medium, which was reconstituted fish water containing AG 828A at concentrations of 0, 100, and 1000 µg·Led as in .−1 AG 828A, and 1 µmol·L−1 tunicamycin  as a positive control. Exposure groups were prepared in HBSS buffer , and a solvent concentration of 0.5% DMSO . Tunicyamycin  is a known ER stress inducer and was furthermore found to induce CYP1A [The isolation of liver of seven adult female zebrafish, followed by a subsequent 5 h exposure to compounds was performed according to . Female ce CYP1A . Afterwace CYP1A . For datTotal RNA of Huh7 cells was isolated using TRIZOL  according to the manufacturer’s instructions. The dried RNA pellet was resuspended in 20 µL of RNAse free water . Total RNA from zebrafish tissue (liver or zebrafish eleuthero-embryos) was extracted using RNeasy Mini Kit  according to the manufacturer’s instructions. RNA concentrations and purity were measured spectrophotometrically , and single strand cDNA synthesis and RT-qPCR were performed as previously described [All data were processed with GraphPad Prism software  and statistics were performed using One-way ANOVA (with Dunett post test).


More recently, a genome-wide association study detected the same genomic region for muscle fatty acid composition in an Iberian x Landrace backcross population. ELOVL6, a strong positional candidate gene for this QTL, contains a polymorphism in its promoter region (ELOVL6:c.-533C < T), which is associated with percentage of palmitic and palmitoleic acids in muscle and adipose tissues. Here, a combination of single-marker association and the haplotype-based approach was used to analyze backfat fatty acid composition in 470 animals of an Iberian x Landrace F2 intercross genotyped with 144 SNPs (single nucleotide polymorphisms) distributed along SSC8.Fat content and fatty acid composition in swine are becoming increasingly studied because of their effect on sensory and nutritional quality of meat. A QTL (quantitative trait locus) for fatty acid composition in backfat was previously detected on porcine chromosome 8 (SSC8) in an Iberian x Landrace FMAML3 and SETD7 are positional candidate genes in the 93 Mb region and two novel microsatellites in MAML3 and nine SNPs in SETD7 were identified. No significant association for the MAML3 microsatellite genotypes was detected. The SETD7:c.700G > T SNP, although statistically significant, was not the strongest signal in this region. In addition, the expression of MAML3 and SETD7 in liver and adipose tissue varied among animals, but no association was detected with the polymorphisms in these genes. In the 119 Mb region, the ELOVL6:c.-533C > T polymorphism showed a strong association with percentage of palmitic and palmitoleic fatty acids and elongation ratios in backfat.Two trait-associated SNP regions were identified at 93 Mb and 119 Mb on SSC8. The strongest statistical signals of both regions were observed for palmitoleic acid (C16:1(n-7)) content and C18:0/C16:0 and C18:1(n-7)/C16:1(n-7) elongation ratios. MAML3 and SETD7 are not the causal mutations for the QTL in the 93 Mb region. However, the results for ELOVL6 support the hypothesis that the ELOVL6:c.-533C > T polymorphism has a pleiotropic effect on backfat and intramuscular fatty acid composition and that it has a role in the determination of the QTL in the 119 Mb region.Our results suggest that the polymorphisms studied in  One of the main sources of human-consumed meat is pork, which represents more than 40% of the meat produced worldwide . The suc2 Duroc x Large White  available from the Sscrofa10.2 database and conserved with the human MAML3 gene [ENSG00000196782].A 931-bp region of the SETD7 gene, two overlapping fragments of 473 bp and 478 bp were amplified and sequenced. Primers [See Additional file Sus scrofa mixed breed [ENSSSCG00000030396] available from the Sscrofa10.2 database and conserved with the human SETD7 gene [ENSG00000145391].For the All primers were designed using the PRIMER3 software  and were2 depending on the primers [See Additional file PCR (polymerase chain reactions) were carried out in a total volume of 25 μL containing 0.6 units of AmpliTaq Gold (Applied Biosystems), 1.5 to 2.5 mM MgClMAML3 gene, two new microsatellites were identified. Both microsatellites were independently amplified using fluorescent primers [See Additional file 2, 0.5 μM of each PCR primer and 0.6 units of AmpliTaq Gold (Applied Biosystems). PCR were run as follows: 94°C for 10 min, 35 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 1.5 min and a final extension step at 72°C for 7 min. The two amplicons were mixed at a ratio of 1:3 (HEX:FAM) and analyzed using capillary electrophoresis on an ABI Prism 3730 DNA analyzer (Applied Biosystems) and the ROX-500 GeneScan Size Standard. The peak height of each product was determined using Peak Scanner 2 software (Applied Biosystems).Based on the sequencing results of the promoter region and exon 1 of the Total RNA was extracted from liver and BF tissues using the RiboPure kit (Ambion), according to the manufacturer’s recommendations. RNA was then quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop products) and RNA integrity was assessed with an Agilent Bioanalyzer-2100 (Agilent Technologies). One μg of total RNA of each sample was reverse-transcribed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) in a reaction volume of 20 μL.MAML3 and SETD7 was analyzed using the 48.48 microfluidic dynamic array IFC chip (Fluidigm) according to the manufacturer’s instructions. Briefly, 2 μL of 1:5 diluted cDNA was pre-amplified using 2X Taqman PreAmp Master Mix (Applied Biosystems) and 50 nM of each primer pair in 5-μL reaction volume. The cycling program consisted of an initial step of 10 min at 95°C followed by 16 cycles of 15 s at 95°C and 4 min at 60°C. At the end of this pre-amplification step, the reaction products were diluted 1:5 (diluted pre-amplification samples). RT-qPCR on the dynamic array chips was conducted on the BioMarkTM system (Fluidigm). A 5-μL pre-mix sample containing 2.5 μL of SsoFast EvaGreen Supermix with Low ROX (Bio-Rad), 0.25 μL of DNA Binding Dye Sample Loading Reagent (Fluidigm) and 2.25 μL of diluted pre-amplification samples , as well as a 5-μL assay mix containing 2.5 μL of Assay Loading Reagent (Fluidigm), 2.25 μL of DNA Suspension Buffer (Teknova) and 0.25 μL of 100 μM primer pairs  were mixed inside the chip using the IFC controller MX (Fluidigm). The cycling program consisted of an initial step of 60 s at 95°C followed by 30 cycles of 5 s at 96°C and 20 s at 60°C. A dissociation curve was also drawn for each primer pair.Fifty-six females of the BC1_LD were used to quantify gene expression. The expression of ACTB, B2M, HPRT1, TBP), ACTB and TBP had the most stable expression [Data were collected using the Fluidigm Real-Time PCR analysis software 3.0.2 (Fluidigm) and analyzed with the DAG expression software 1.0.4.11  using stpression  in both PCR primer sequences [See Additional file Mean values between genotypes were compared using a linear model implemented in R, which performs a single stratum analysis of variance considering sex and batch as fixed effects. Differences were considered statistically significant at a p-value of 0.05.2 animals. Association analyses for the BF FA composition in the C14:0 to C22:0 ranges were performed with genotypes from a subset of 133 SNPs . Statistically significant associations were found (Table A custom panel of 144 SNPs located on SSC8 was used to genotype 470 FTwo regions that contain trait-associated SNPs (TAS) were clearly visualized in the association plots at around 93 Mb and 119 Mb for all of the above-mentioned FA and indices with the exception of the C20:2(n-6)/C18:2(n-6) elongation ratio, which showed only one significant TAS region at 120.99 Mb  was then performed for the most significantly associated traits [See Additional file In order to determine whether one or two QTL were segregating on SSC8 for the BF FA and their indices, models fitting one QTL against a model considering two different QTL were tested. Results of the LR test indicated that the model with two QTL was the most likely for the 10 traits analyzed [See Additional file 2 generation [FABP2[MTTP[2 cross [2 cross [Previously, a QTL scan for BF FA composition was performed with 369 animals from the same Fneration , but onlon [FABP2 and MTTPABP2[MTTP, but the[2 cross  and for [2 cross .Sscrofa10.2 assembly are located at 99.2 Mb and 119.7 Mb, respectively. Furthermore, two significant pleiotropic regions (at 93.3 Mb - 99.5 Mb and 110.9 Mb - 126.9 Mb) with effects on palmitoleic (C16:1(n-7)) FA in both IMF and BF tissues have been identified in the same backcross [A GWAS for IMF FA composition  with genackcross . For palackcross .2 animals. The palmitoleic (C16:1(n-7)) FA QTL on SSC8 have been shown to be segregating in different crosses of the IBMAP population, and both QTL have a pleiotropic effect on BF and IMF FA deposits.Here, two QTL at approximately 93 Mb and 119 Mb were detected and affected the BF composition of the six FA and the four indices mentioned above in the 470 Fmastermind-like 3 (MAML3) (at position 92.67 Mb) and SET domain containing lysine methyltransferase 7 (SETD7) (at position 93.13 Mb) were found. Both genes have recently been reported in a predicted co-association gene network for intramuscular FA composition in pigs .Gene annotation of the two TAS genomic regions allowed us to identify genes related to FA metabolism. In the first region, the genes MAML3 is a member of the Mam gene family, which plays an essential role in the stabilization of Notch transcriptional activation complexes [MAML3 gene that covers part of the promoter region and part of exon 1, was amplified from genomic DNA and sequenced. Two novel microsatellites were found: MAML3_MS1, a (CA)n tandem repeat located in the promoter region and MAML3_MS2, a (CGG)n tandem repeat identified in exon 1. The variability of both microsatellites is described in Table omplexes . This Noomplexes . A 931-bSETD7 gene is a histone methyltransferase that specifically monomethylates Lys-4 of histone H3 [collagenase or insulin[SETD7 gene, a 839-bp fragment of the SETD7 promoter and exon 1 was amplified from genomic DNA and sequenced. In addition, the identification of polymorphisms in the entire coding region of the SETD7 gene was performed using RNA-Seq data [http://www.broadinstitute.org/igv/). Alignment and analysis of these sequences led to the identification of nine polymorphisms (Table SETD7:c.-1034T > G) and one non-synonymous polymorphism in exon 6 (SETD7:c.700G > T), which determines an amino-acid change of valine to leucine. Apart from the fact that these SNPs are located in the SETD7 gene, they were selected because they showed divergent allelic frequencies between the Iberian and Landrace IBMAP founders i.e. the SETD7:c.-1034 T and SETD7:c.700 T alleles were fixed in the Iberian boars. Complete linkage disequilibrium between the two SNPs was observed in the genotyped BC1_LD animals and, thus, only SETD7:c.700G > T was further genotyped in 168 animals belonging to the F2 generation.The product of the stone H3  and, thur insulin. To idenSeq data  with theELOVL6 gene was identified at position 120.12 Mb. The ELOVL6 gene is a strong positional and functional candidate gene involved in de novo lipogenesis and acts on the elongation of SFA and MUFA. A polymorphism in the promoter region of this gene (ELOVL6:c.-533C > T) has previously been associated with percentages of palmitic and palmitoleic FA in muscle and backfat in the BC1_LD population [ELOVL6 gene was lower in the backfat of animals with the Iberian allele in comparison to those with the Landrace allele. As expected from the elongation function of this gene, a lower ELOVL6 expression was associated with a higher percentage of palmitic and palmitoleic FA in muscle and adipose tissue [In the second region, the pulation . In addie tissue .ELOVL6 gene in animals with the Iberian allele [ELOVL6 elongates palmitic (C16:0) to stearic (C18:0), and palmitoleic (C16:1(n-7)) to vaccenic (C18:1(n-7)) FA. Thus, a lower ELOVL6 activity associated with the Iberian allele will directly decrease these elongation ratios. Moreover, as observed, a lower ELOVL6 activity will result in the accumulation of palmitic (C16:0) and palmitoleic (C16:1(n-7)) FA and a reduction in stearic (C18:0) FA content ), the main dietary FA, its content in BF was decreased in animals with the Iberian allele for the TAS regions on SSC8. It must be noted that the opposite effect was observed in the major SSC4 and SSC6 TAS regions for oleic (C18:1(n-9)) IMF content .MAML3 and SETD7 genes were studied in liver and BF tissues of 56 BC1_LD females by RT-qPCR. The analysis of gene expression in the F2 generation was not possible because tissues for RNA isolation were not available. Differences in the expression of MAML3 among animals were observed, with coefficients of variation (CV) of 35% and 42% in liver and BF, respectively. The SETD7 gene expression was less variable, with CV values of 18% and 33% in liver and BF tissue, respectively. However, no significant differences in expression of SETD7 were detected among animals classified according to the SETD7 genotypes (either SETD7:c.-1034T > G or SETD7:c.700G > T) in either tissue. Similarly, no differences in expression of MAML3 were observed among animals classified according to the MAML3_MS1 and MS2 microsatellites. In addition, no significant correlation was found between expression levels of MAML3 or SETD7 in the liver and adipose, which suggests that different and tissue-specific mechanisms control the liver and adipose tissue expression of MAML3 and SETD7.The expression profiles of the pig MAML3 gene (MAML3_MS1 and MAML3_MS2), one SNP in the SETD7 gene (SETD7:c.700G > T), and one SNP in the ELOVL6 gene (ELOVL6:c.-533C > T) were used to genotype 168 animals of the F2 generation. An association analysis with these markers and the SSC8 genotypes from the 133 SNPs of our custom porcine SNP panel was performed.Two microsatellites in the SETD7 and MAML3 genes were studied. For SETD7, the SETD7:c.700G > T polymorphism did not show the most significant association , polymorphisms in the on Table . In addils Table  were locELOVL6 gene was studied. The ELOVL6:c.-533C > T polymorphism showed the highest association with percentage of palmitic and palmitoleic FA, ACL, and C18:0/C16:0 and C18:1(n-7)/C16:1(n-7) ratios (Table ELOVL6:c.-533C > T polymorphism with percentage of FA in IMF and BF indicates a pleiotropic effect of this gene in both tissues.For the second region (119 Mb), a polymorphism in the os Table . Hence, SETD7:c.700G > T and ELOVL6:c.-533C > T showed a higher contribution of ELOVL6:c.-533C > T SNP for all studied FA and indices. Furthermore, the additive value of the two SNPs [See Additional file Analysis of the additive value of SNPs MAML3 and SETD7 genes were analyzed as positional candidate genes of the 93-Mb TAS region. Two novel microsatellites were identified in the MAML3 gene, and nine SNPs in the SETD7 gene. However, the association analysis did not reveal any significant association between the MAML3 microsatellite genotypes and the traits studied, and the SETD7:c.700G > T SNP did have not the strongest signal in the 93 Mb region. Although the expression of MAML3 and SETD7 genes in liver and adipose tissue varied among animals, it was not associated with any of the genotyped polymorphisms in these genes. These results suggest that the polymorphisms studied in MAML3 and SETD7 are not the causal variants of the 93-Mb QTL. Conversely, for the 119-Mb region, the ELOVL6:c.-533C > T SNP was strongly associated with percentage of palmitic and palmitoleic FA, ACL, and C18:0/C16:0 and C18:1(n-7)/C16:1(n-7) elongation ratios. These results suggest pleiotropic effects of ELOVL6:c.-533C > T on BF and IMF FA composition.In summary, two TAS regions at 93 Mb and 119 Mb on SSC8 affect BF FA composition. Both regions showed a strong effect on palmitoleic acid content and C18:0/C16:0 and C18:1(n-7)/C16:1(n-7) elongation ratios. The The authors declare that they have no competing interests.JMF, MB and YRC conceived and designed the experiment. JMF was the principal investigator of the project. NI, MM and JMF collected samples. APO, JC, MB and MR performed the DNA and RNA isolation. MR, AC, JC and MB identified the polymorphisms and performed the genotyping. MR, YRC and JC performed the association analysis. MR, AC, APO and MB performed the gene-expression analysis. MR and JMF wrote the manuscript. All authors read and approved the final manuscript.List of SNPs genotyped. List of 144 SNPs located on SSC8 genotyped and genotyping statistics.Click here for fileSETD7 and MAML3 promoter sequencing (P), promoter and exon 1 sequencing (PE) and microsatellite genotyping (MS). Table S3. Significant SNPs affecting BF FA composition (FDR = 0.05) in LDLA analyses in the F2 generation. Table S4. Analysis of a two QTL model on SSC8 for the most significant regions affecting BF FA composition. Table S5. Additive value affecting BF FA composition in 168 F2 animals for the SETD7:c.700G > T and ELOVL6:c.533C > T SNPs.Primers for Click here for fileMAML3 gene, the blue line represents the position of the SETD7 gene and the green line represents the position of the ELOVL6 gene on SSC8; horizontal, dashed ines mark the association study significance level ; positions in Mb are relative to the Sscrofa10.2 assembly of the pig genome.Association study and LDLA of the C16:1(n-7), ACL and C18:1(n-7)/C16:1(n-7) elongation ratio in BF. Plot of association study (red points) and LDLA patterns (blue line) for palmitoleic acid, ACL and vaccenic/palmitoleic ratio; the X-axis represents positions in Mb on SSC8, and the Y-axis shows the –log10 ; vertical, the pink line represents the position of the Click here for file


The genetic polymorphism concerning the ß3-subunit of platelet integrin receptor glycoprotein IIIa is held responsible for enhanced binding of adhesive proteins resulting in increased thrombogenic potential. Whether it is associated with mortality, HbA1c or platelet volume is tested prospectively in an epidemiological cohort.1c, and mean platelet volume. Multivariate analysis was performed to investigate association between genotype and key variables.Population-based Cooperative Health Research in the Region of Augsburg (KORA) S4-Survey  was investigated for prognostic value of PLA1A2-polymorphism regarding all-cause mortality, correlation with HbA1c was a prognostic marker of mortality in non-diabetic persons resulting in J-shaped risk curve with dip at HbA1c = 5.5% (37 mmol/mol), confirming previous findings regarding aged KORA-S4 participants (55–75 years). PLA1A2 was significantly associated with elevated HbA1c levels in diabetic patients (N = 209) and reduced mean platelet volume in general population. In non-diabetic participants , carriers of PLA2 allele variant presenting with HbA1c > 5.5% (37 mmol/mol) showed higher relative risk of mortality with increasing HbA1c.Prevalence of thrombogenic allele variant PLA2 was 15.0%. Multivariate analysis revealed no association between PLA1A2 polymorphism and mortality in the KORA-cohort. HbA1c ranging from 5.5% (37 mmol/mol) to 6.5% (48 mmol/mol). Maintenance of euglycemic control and antiplatelet therapy are therefore regarded as effective primary prevention in this group.PLA1A2 polymorphism is associated with mortality in participants with HbA Platelets play an important role in primary hemostasis and are involved in atherosclerosis and atherothrombotic events. Inhibition of platelet aggregation is the key step of any treatment of vascular disease. Metabolic conditions like hyperglycemia influence platelet reactivity and the response to platelet inhibitors through direct effects and by glycation of platelet proteins, especially in type 2 diabetes. Increased platelet reactivity involves intensified adhesion and aggregation in patients with diabetes mellitus or those at high risk for the disease. A subpoIn a study on Japanese subjects MPV in patients with prediabetes was shown to be higher if compared to normal subjects, and it was positively associated with levels of fasting blood glucose in prediabetic and normal subjects. In a KoITGB3 rs5918)] at position 1565 in exon 2 of the coding region for glycoprotein IIIa and results in a leucine-proline exchange[in vitro[Numerous polymorphic surface glycoprotein receptors are responsible for platelet functionality with membrane glycoprotein (GP) IIb/IIIa playing a major role in platelet function. It enables stimulated platelets to bind to fibrinogen and related adhesive proteins, a process that is considered central in the development of thrombosis. The gene encoding GPIIIa shows a common platelet antigen polymorphism [PLA1A2,  its relation with main characteristics of platelet morphology.With this regard, the aim of this prospective analysis of the KORA S4-survey is to clarify 1) the predictive role of the PLA1A2 polymorphism in the general population in terms of all-cause mortality, 2) its relation with HbA1c levels at baseline ≥6.5% (48 mmol/mol) (N = 209 participants)[1c was determined centrally at baseline[The KORA study region consists of the city of Augsburg and the two surrounding districts with about 600,000 inhabitants in 1999. The Bavarian ethic committee approved the KORA S4 study (conducted between 1999 and 2001) which followed the declaration of Helsinki; informed consent was given by each participant. The initial study sample involved 6,640 subjects randomly drawn from the general population. Altogether 4,261 subjects participated in the baseline study (response 67%). Of those, 4,028 had been characterized according their PLA1A2 polymorphism by a flow cytometry based assay as described elsewhere and coulcipants). HbA1c wDescriptive analysis results of the population characteristics were reported as mean ± standard deviation (SD). Comparison between the groups was done by Mann–Whitney testing or one-way ANOVA followed by Dunnett’s multiple comparison post-test for continuous data and Fisher’s exact test for categorical data.1c, MPV, platelet mass and platelet count. Variables investigated for possible confounding included age, sex, BMI, waist-hip ratio, diastolic and systolic blood pressure, cholesterol levels , smoking status , high alcohol intake , leisure time physical activity (categorized: >1 h per week). Association between platelet count and covariates were investigated by linear regression model.A multivariate logistical regression model was used to evaluate the cross-sectional association of genotype with HbA1c level and platelet morphology , taking the same adjustment as previously described. Statistical analysis was done using R version 2.15.1 . P values <0.05 were regarded statistically significant.Cox proportional hazards model was used for a multivariate analysis of the risk of overall death with genotypes, HbA1c, PLA1A2 genotype was determined by flow cytometry and data on the survival status were available. Total prevalence of the PLA2 allele was 15.0%, genotype distribution was as follows: A1A1: 2,912/4,028 = 72.3%, A1A2: 1,027/4,028 = 25.5%, A2A2: 89/4,028 = 2.2%. For evaluating the role of the PLA1A2 genotype two groups were set up comprising A1A1 genotypes and A1A2/A2A2 genotypes, called AxA2. We detected no significant difference in prevalence of AxA2 genotype in the living and deceased participants  than in AxA2 (11%), 40% of A1A2 participants received oral antidiabetics only, whereas in the AxA2 group this therapeutic regimen was followed by 38%, the remaining participants in the groups did not receive any antidiabetic medication. Participants with diabetes significantly received more often acetylsalicyl acid than non-diabetics (25.0% vs 5.3%), but the application of acetylsalicyl acid did not differ between the groups A1A1 and AxA2. Regarding antidiabetic and antithrombotic therapy a possible confounding between the groups A1A2 and AxA2 therefore is not to be expected.Mean diabetes duration did not differ statistically significant in both groups (A1A1 and AxA2) .1c ≥6.5% (48 mmol/mol), N = 209), an association between PLA1A2 and elevated HbA1c levels could be found  after multivariable adjustment , BMI , waist-hip-ratio  were significantly associated with MPV in multivariate linear regression model.A significant association between genotype AxA2 and lower MPV was demonstrated in the whole cohort and the group of non-diabetic people, but there was no significant association for platelet mass and platelet count  =1.17 ; HbA1c: adjusted HR (95% CI) =1.05 ) regarding all participants.Within 10 years 239 (5.9%) patients died: 167 (5.7%) in the A1A1, 72 (6.5%) in the AxA2 (68 (6.6%) in the A1A2 and 4 (4.5%) in the A2A2) group, mainly due to cardiovascular diseases like myocardial infarction and ischemic heart disease (49%) as well as cancer (39%). No significant impact of genotype or HbA1c, genotype and platelet morphology  in the non-diabetic participants (HbA1c < 6.5% (48 mmol/mol)), we confirmed HbA1c = 5.5% (37 mmol/mol), which was reported by Kowall et al. regarding KORA S4 participants aged from 55 to 75 years[1c level and risk of all-cause mortality, but PLA1A2 polymorphism was not significantly associated with overall cause of death in this group , but was not related to survival in subjects with HbA1c ≤ 5.5% (37 mmol/mol) (Table 1c ≤ 5.5% (37 mmol/mol) platelet count and platelet mass, but not MPV were associated with all-cause mortality  in separated genotypes to investigate possible genotype-specific effects (Table 1c > 5.5% (37 mmol/mol) up to <6.5% (48 mmol/mol) a significant positive relation was found between HbA1c level and all-cause mortality only in people with AxA2 genotype (Table 1c and AxA2 genotype in this subgroup. For the participants with HbA1c ≤ 5.5% (37 mmol/mol), HbA1c was not significantly associated with mortality ) (Table 1c < 5.5% (37 mmol/mol), platelet count, platelet mass and MPV were associated with the all-cause mortality  as reference is presented in Figure 1c = 5.5% (37 mmol/mol) to higher HbA1c, while in participants with genotype A1A1, the increase was much less pronounced. In the highest decile (HbA1c > 6.0% (42 mmol/mol)), the adjusted hazard ratio (95% CI) was 2.97  in people with genotype AxA2 compared to 1.35  in people with A1A1.The hazard ratios of all-cause mortality in each decile of HbA1c and mortality in non-diabetic subjects and confirms previously published results regarding older participants of KORA S4.Atherosclerosis results from complex interactions between the environment and genetic factors. Individual hemostatic platelet response may be influenced by the genetic profile of the platelet membrane glycoprotein (GP) receptors. As part of von Willebrand factor and fibrinogen receptor GPIIIa plays a pivotal role in platelet aggregation. Numerous polymorphisms in platelet surface glycoproteins have received particular interest; one of The role of GPIIIa polymorphism in genetic susceptibility to clinical thrombotic disease still remains controversial,19. The 1c (HbA1c > 5.5% (37 mmol/mol) to <6.5% (48 mmol/mol)) including prediabetic subjects, people with PLA2 SNP showed higher mortality risk for same increase of HbA1c level compared to persons with HbA1c ≤ 5.5% (37 mmol/mol), pointing to a combined effect of HbA1c and AxA2 genotype in this subgroup. This effect was not detected in A1A1 subjects. HbA1c had no significant influence on survival in the diabetic patients of KORA; this might be due to the limited number of subjects with elevated HbA1c in this cohort.Our data do not suggest an overall linkage between PLA1A2 genotype and all-cause mortality but put further substance to the assumption that the PLA2 SNP is associated with impaired metabolic control in diabetes mellitus. In the group of non-diabetics with higher HbA1c turned out to be a strong predictor for survival in the non-diabetic population of the KORA cohort with the lowest risk at a baseline level of 5.5% (37 mmol/mol). This finding is in line with a previous publication by Kowall et al. regarding KORA S4 participants aged from 55 to 75 years[1c with overall mortality are seen in population studies without diabetes like the ARIC (Atherosclerosis Risk in Communities) study[1c values and optimal HbA1c with lowest mortality events around 7.5%[1c may be attributable to the level of comorbidities[HbA75 years. Within 75 years-37. Howe75 years-41. Thess) study and Natiund 7.5%; the higbidities.1c, and suffer higher relative risk of mortality than people with A1A1 genotype for the same increase of HbA1c level in the range of 5.5% (37 mmol/mol) to 6.5% (48 mmol/mol), resembling a prediabetic metabolic situation. The association of HbA1c and all-cause of death was PLA1A2 genotype specific. At each decile the curve of AxA2 carriers is shifted to elevated hazard ratios, while in people with A1A1 the HbA1c level was not significantly associated with mortality . This fact clearly points to the role of platelet characteristics in the group of definite non-diabetics, contributing to survival/mortality. In this cohort subjects with AxA2 genotype present with significant lower MPV but preserved platelet mass, which might be an indicator of increased platelet consumption, mainly of the larger platelet-subpopulation that present more receptors per platelet[An important additional finding on survival prediction in our analysis is that MPV, platelet count and platelet mass are significantly associated with survival in the individuals with HbAplatelet. Due to 1c values of >5.5% (37 mmol/mol) up to 6.5% (48 mmol/mol), including the prediabetic subjects. Therefore, even the prediabetic subject has to be regarded as a vulnerable vascular patient, which has recently been confirmed by the Silent Diabetes Study, published by Doerr and co-authors[PLA2 significantly correlates with mortality in non-diabetics with HbA-authors. Elevate-authors. Our resKORA: Kooperative Gesundheitsforschung im Raum Augsburg ; GP: Glycoprotein; GWAS: Genome wide association studies; MPV: Mean platelet volume; FBG: Fasting blood glucose; QTL: Quantitative trait locus; MAF: Minor allele frequency.The authors declare that they have no competing interests.All authors had access to the final data, the manuscript and accept the responsibility for its validity. The authors declare that there is no conflict of interest. BS literature search, data analysis, data interpretation, writing of the manuscript. TX data analysis, data interpretation, writing of the manuscript, design of figures. CM data collection, data interpretation, BM data collection, CH data collection, MR data handling, HG data collection and handling. AP data analysis, design of figures. WK data collection. TI data collection and handling. HW data collection. RWS data analysis. WR data collection. DT data collection, data interpretation, writing of the manuscript. All authors had access to the final data, the manuscript and accept the responsibility for its validity.


Enterobacter ludwigii DSM 16688T within the Enterobacteriaceae family, although these isolated bacteria showed slightly different morphology before and after Cu shock load. Two representative strains R2B1 (before Cu shock load) and B4B2 (after Cu shock load) were chosen for further analysis. B4B2 is resistant to 200 mg L−1 of Cu(II) while R2B1 is not, which indicated the potential selection of the Cu shock load. Raman analysis revealed that both R2B1 and B4B2 contained c-type cytochromes. Cyclic voltammetry measurements revealed that strain R2B1 had the capacity to transfer electrons to electrodes. The experimental results demonstrated that strain R2B1 was capable of utilizing a wide range of substrates, including Luria-Bertani (LB) broth, cellulose, acetate, citrate, glucose, sucrose, glycerol and lactose to generate electricity, with the highest current density of 440 mA·m−2 generated from LB-fed MFC. Further experiments indicated that the bacterial cell density had potential correlation with the current density.Microorganisms capable of generating electricity in microbial fuel cells (MFCs) have gained increasing interest. Here fourteen exoelectrogenic bacterial strains were isolated from the anodic biofilm in an MFC before and after copper (Cu) shock load by Hungate roll-tube technique with solid ferric (III) oxide as an electron acceptor and acetate as an electron donor. Phylogenetic analysis of the 16S rRNA gene sequences revealed that they were all closely related to  In a microbial fuel cell (MFC), electroactive microorganisms are capable of generating electricity directly from organic compounds. Due to their specific ability to transfer electrons outside the cell to the anode of the MFC, these bacteria are renowned as exoelectrogens (“exo-” for extracellular and “electrogens” for the ability) Geobacter sp., Shewanella sp., Pseudomonas sp., Burkholderia sp., Escherichia coli, Ochrobactrum anthropi, Rhodoferax ferrireducens, Citrobacter sp. and Clostridium sticklandii  2) in the anaerobic serum bottle (100 ml) containing 50 ml of sterile anaerobic culture medium. The isolation was performed by serial dilution using Hungate roll-tube technique with the basal medium (BM) 4Cl, 0.026 g KH2PO4, 1.64 g sodium acetate, 0.2 g yeast extract (Oxoid), 0.5 g cysteine hydrochloride, 1 mg resazurin (Sigma), 12.5 ml mineral and 12.5 ml vitamin solutions per liter . The electron acceptor was 20 mM amorphous Fe(III) oxyhydroxide, which was synthesized by neutralizing a 0.4 M FeCl3 solution to a pH of 7 with 1.2 M NaOH 2-free N2 gas and cooled to room temperature. Five-mililiter of the medium was dispensed into Hungate tubes and 50 ml medium was dispensed into serum bottles. The sealed vessels were then autoclaved for 20 min at 121°C. The culture was purified by using a repeated Hungate roll-tube method with BM agar. Single black colonies were picked out and inoculated into the same liquid medium.A continuous-flow MFC was constructed to contain three rectangular chambers , which were separated by Plexiglas baffle without conventional proton exchange membrane (PEM). Substrate utilization of isolated strains was determined by conventional methods using Biolog GN2 MicroPlates . Enzyme activity was carried out on API ZYM (bioMérieux) according to the manufacturer's instructions.5′-GGTTACCTTTGTTACGACTT-3′) To confirm the purity and the similarity of the isolated strains, genomic DNA was extracted using the FastDNA SPIN Kit for Soil  according to the manufacturer's instructions. The 16S rRNA gene was PCR amplified using the primer 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R  was also used for comparison in the assay A series of LB agar containing different Cu(II) concentrations, including 0, 50, 100, 150, 175, 200, 250, 300 to 400 mg LThe morphologies of bacterial cells growing overnight in anaerobic tubes were examined with a scanning electron microscope (SEM) . For SEM, the suspended cells were fixed in a 2.5% glutaraldehyde and 0.1 M phosphate buffer solution  for 2 h, and then dehydrated with a graded ethanol series from 30 to 100% for 20 min each. After dehydration, the samples were critical-point dried and then sputter-coated with gold under vacuum for SEM examination A 5 µl cell suspension was placed on 200 mesh Formvar carbon-coated copper grid and wicked off after 3 min. The sample was soaked in 5 µl of uranyl acetate (2%) for 30 s, then drained and air-dried and examined using a transmission electron microscopy (TEM)  at an accelerating voltage of 80 kV.Cyclic voltammetry (CV) was performed using a potentiostat  to investigate the electron transfer mechanism. The CV experiment was conducted with a three-electrode system with glassy carbon electrode as working electrode, Ag/AgCl as reference electrode, and a platinum wire as counter electrode according to a previous study −1. Spectral acquisition was done at integration times of 0.2 s to obtain high-contrast resonance spectra for the c-type cytochromes.To perform confocal Raman microscopy, the anodes were cut from the incubation vessels and transferred immediately into a sterile cell culture dish filled with 50 ml of PBS. Raman spectra were acquired using a confocal micro-Raman system  equipped with a Leica microscope, charge coupled device detector. Excitation was provided by a He-Ne 532 nm laser with the power of 5 mW on the sample. Raman signals were collected with a 50 mm optical fibre with a resolution of 4 cmEnterobacter strain R2B1 using various substrates including citrate, acetate, glucose, sucrose, glycerol, lactose, cellulose and LB medium in a fed-batch mode in an incubator (30±2°C). Pure cellulose was microcrystalline insoluble cellulose, 15% amorphous and 85% crystalline with a 50-mm particle size . The MFCs were made of Plexiglas and anodic and cathodic compartments had an internal dimension of 5×5×5 cm, which were separated by a PEM . The PEM was pretreated by boiling it in 3% hydrogen peroxide for 1 h, and subsequently in 1.0 M sulfuric acid for another 1 h in boiling deionized water, and then stored in deionized water before use. Carbon felt with a surface of 3×3 cm was used as the electrode material. The anode solution (100 ml) was modified based on 0.05 M PBS (pH = 7.4) containing 0.12 g NH4Cl, 8 g NaCl, 0.13 g KCl, 7.8 g NaH2PO4·2H2O, 17.9 g Na2HPO4·12H2O, and 12.5 ml mineral and 12.5 ml vitamin solutions per liter 3Fe(CN)6] in PBS . MFCs were inoculated with 3 ml late exponential phase cultures of isolated bacteria grown in the LB broth.Two-chamber MFCs were constructed for evaluation of power generation by the isolated −2)  = 10×V2/(R×A), where V (mV) is the output voltage, A (cm2) is the surface area of the anode, and R (Ω) is the external resistance 2) of the cathodic electrode.Cell voltage (V) was recorded online every 10 min via a data acquisition system  which was connected to a personal computer. The current (I) was calculated from the fixed external resistance (R = 1 kΩ) and cell voltage (V) according to Ohm's law (I = V/R). Power (P) was calculated by using P  shock load, two representative strains R2B1 (before Cu shock load) and B4B2 (after Cu shock load) were chosen for further analysis. Strains R2B1 and B4B2 are gram-negative and the SEM and TEM images illustrated that they are 0.5–0.6 µm wide and 0.7–1.8 µm long rod, with a polar flagellum  of isolated strains were obtained and subjected to comparative analysis with the 16S rRNA genes of closely related reference strains. According to the lagellum . The cel of B4B2 . In addiEnterobacter sp. R2B1 and B4B2 were isolated from a MFC system which was exposed before and after a Cu shock load, the growth of these two strains under a series of different Cu(II) concentrations, ranging from 0 to 400 mg L−1, was conducted to investigate the Cu resistance of these two strains  concentration, while R2B1 could not. In addition, it was found that strain B4B2 grew more rapidly compared with strain R2B1 on the LB agar plate in the presence of Cu(II). These results implied that the Cu shock load promoted the selection of Cu resistant exoelectrogens.Since  strains . Even thEnterobacter sp. strains R2B1 and B4B2 to use various carbon sources are shown in Enterobacter cloacae FR could produce electricity in an MFC. Therefore, selective carbon sources utilized by Enterobacter cloacae FR and the type strain Enterobacter cloacae ATCC 13047T were compared with strains R2B1 and B4B2. As shown in E. cloacae FR nor E. cloacae ATCC 13047T could use these carbon sources.Biochemical characterization of For the API ZYM enzyme assay, nine enzymes were produced by both of the isolates, including phosphatase, leucine aminopeptidase, valine aminopeptidase, cystine aminopeptidase, trypsin, acid phosphatase, β-galactosidase, α-glucosidase and n-acetyl-β-glucosaminidase . DiffereEnterobacter sp. strain R2B1. The oxidation peak corresponding to −0.198 V (vs Ag/AgCl) was likely due to the c-type cytochromes To further investigate the electrochemical characteristics, CV analysis of strain R2B1 on glassy carbon were conducted using a three-electrode system in 50 mM deoxygenated PBS solution. As shown in c are at high densities in electroactive bacteria as they play significant roles in dissimilatory reduction of Fe and Mn oxides c was used as the indicator for the characterization of an electroactive biofilm using Raman microscopy c produced strong, representative and reproducible Raman spectra when excited with a 532 nm wavelength due to the enhancement of the Raman scattering (Raman resonance effect) of the porphyrin ring in visible and ultra-violet bands c. According to the results  were observed for the two isolated strains, indicating the potential high densities of cytochrome c in them. Previous studies reported that cytochrome c associated with bacterial outer membrane and conductive pili could be used for direct electron transfer Enterobacter aerogenes biofilms Certain proteins, such as cytochromes, are crucial in electron transfer via the physical contact of the bacterial cell membrane. It has been demonstrated that cytochrome  results , four ty−2 at the fixed resistance, presumably due to higher substrate concentrations from tryptone and yeast extract, followed by citrate, cellulose and acetate with 144±6.3 mA·m−2, 118±7.3 mA·m−2 and 93±0.9 mA·m−2, while the maximum current densities of other substrates were rather low (less than 100 mA·m−2). These results illustrated that the strain R2B1 could utilize a wide range of substrates for electricity generation in MFCs but with different power generation abilities. Although strain R2B1 in this study and the Enterobacter cloacae ATCC 13047T reported by Razaei Enterobacter, they display dissimilarity in terms of electricity production from substrates. For instance, strain R2B1 could oxidize acetate for electricity generation, while E. cloacae ATCC 13047T could not.The isolated strain R2B1 was used to determine its ability to generate current from a variety of substrates, including glucose, lactose, sodium acetate, glycerol and sucrose with an external resistance of 1 kΩ . The LB −2, indicating that LB medium are most suitable for the growth of strain R2B1's anode biofilm. DNA contents of the anode samples from the MFCs fed with citrate, acetate and cellulose were 281, 208 and 164 mg m−2, respectively. There was no significant differences among glucose (130 mg m−2), sucrose (115 mg m−2) and glycerol (150 mg m−2) fed MFCs. In general, the bacterial cell density was similar to the trends observed with the current density Click here for additional data file.Figure S2The photographs of the isolated strains cultivated on LB agar plates with different concentrations of copper ions.(TIF)Click here for additional data file.Figure S3Images of electroactive biofilms using LB as the carbon source. Green indicates the electrochemically active biofilm. Black indicates carbon electrode.(TIF)Click here for additional data file.Table S1Taxa of exoelectrogens.(DOC)Click here for additional data file.Table S2Growth on various electron donors of Enterobacter sp. strain R2B1 and B4B2.(DOC)Click here for additional data file.