De novo design provides an attractive approach, which allows one to test and refine the principles guiding metalloproteins in defining the geometry and reactivity of their metal ion cofactors. Although impressive progress has been made in designing proteins that bind transition metal ions including iron-sulfur clusters, the design of tetranuclear clusters with oxygen-rich environments remains in its infancy. In previous work, we described the design of homotetrameric four-helix bundles that bind tetra-Zn2+ clusters. The crystal structures of the helical proteins were in good agreement with the overall design, and the metal-binding and conformational properties of the helical bundles in solution were consistent with the crystal structures. However, the corresponding apo-proteins were not fully folded in solution. In this work, we design three peptides, based on the crystal structure of the original bundles. One of the peptides forms tetramers in aqueous solution in the absence of metal ions as assessed by CD and NMR. It also binds Zn2+ in the intended stoichiometry. These studies strongly suggest that the desired structure has been achieved in the apo state, providing evidence that the peptide is able to actively impart the designed geometry to the metal cluster.

Pharmaceuticals in waterbodies are a growing concern due to their extensive uses and adverse effects on aquatic life. Oxytetracycline (OTC) is one of tetracycline antibiotic group used for treatment of animals and humans. This study evaluates the simultaneous oxidation of OTC and ammonium under autotrophic nitrifying conditions by using a membrane aerated biofilm reactor (MABR) as it provides an appropriate environment for the antibiotic-degrading bacteria. The results showed that MABR achieved fluxes of 1.62 mg OTC/m2.d and 1117 mg N/m2.d while the fluxes of O2 (JOTC-O2) utilized for OTC and NH4-N (JNH4-N-O2) oxidation were calculated to be 2.94 and 5105 mg O2/m2.d, respectively. Three transformation products, 4-Epi-OTC, α-Apo-OTC and β-Apo-OTC, were identified and measured at ppb levels. The biofilm community comprised of Bacteria environmental samples, b-proteobacteria, CFB group bacteria, g-proteobacteria, d-proteobacteria and a-proteobacteria.

Lycopene is the red pigment in tomatoes and tomato products and is an important dietary carotenoid found in the human organism. Lycopene-isomers, oxidative lycopene metabolites and apo-lycopenoids are found in the food matrix. Lycopene intake derived from tomato consumption is associated with alteration of lipid metabolism and a lower incidence of cardiovascular diseases (CVD). Lycopene is mainly described as a potent antioxidant but novel studies are shifting towards its metabolites and their capacity to mediate nuclear receptor signalling. Di-/tetra-hydro-derivatives of apo-10´-lycopenoic acid and apo-15´-lycopenoic acids are potential novel endogenous mammalian lycopene metabolites which may act as ligands for nuclear hormone mediated activation and signalling. In this review, we postulate that complex lycopene metabolism results in various lycopene metabolites which have the ability to mediate transactivation of various nuclear hormone receptors like RARs, RXRs and PPARs. A new mechanistic explanation of how tomato consumption could positively modulate inflammation and lipid metabolism is discussed.

Mtb β-lactamase (BlaC) is extremely efficient in hydrolyzing β-lactam antibiotics which renders/leads to protection and/or resistance to this bug. There is a compelling need to develop new non-lactam inhibitors which can bind and inhibit BlaC, but cannot be hydrolyzed, thus neutralizing this survival mechanism of Mtb. Using the crystal structure of BlaC we screened 750000 purchasable compounds from ZINC Database for their theoretical affinity to the enzyme's active site. 32 of the best hits of the compounds having tetra-, tri- and thiadi-azole moiety were tested in vitro, and 4 efficiently inhibited the enzymatic activity of recombinant BlaC. Characterization of the shape of BlaC-/+ inhibitors by small angle X-ray scattering (SAXS) brought forth that BlaC adopts: (1) an open shape (radius of gyration of 2.3 nm compared to 1.9 nm of crystal structures) in solution; (2) closed shape similar to observed crystal structure(s) in presence of effective inhibitor; and (3) a closed shape which opens up when a hydrolysable inhibitor is present in solution. New BlaC inhibitors were: 1-(4-(pyridin-3-yl)-thiazol-2-ylamino)-2-(7,8,9-triaza-bicyclo[4.3.0]nona-1(6),2,4,8-tetraen-7-yl)-ethanone; 8-butyl-3-((5-(pyridin-2-yl)-4H-1,2,4-triazol-3-ylamino)-formyl)-8-aza-bicyclo[4.3.0]nona-1(6),2,4-triene-7,9-dione; 1-(3-((5-(5-bromo-thiophen-2-yl)-1,3,4-oxadiazol-2-yl)-methoxy)-phenyl)-1H-1,2,3,4-tetraazole; and 1-(2,3-dimethyl-phenylamino)-2-(2-(1-(2-methoxy-5-methyl-phenyl)-1H-1,2,3,4-tetraazol-5-ylsulfanyl)-acetylamino)-ethanone. The open-close shape of BlaC questions the physiological significance of the closed shape known for BlaC-/+ inhibitors and paves new path for structure aided design of novel inhibitors.

Although metal ion binding to naturally occurring l-amino acid proteins is well documented, understanding the impact of the opposite chirality (d-)amino acids on the structure and stereochemistry of metals is in its infancy. We examine the effect of a d-configuration cysteine within a designed l-amino acid three-stranded coiled coil in order to enforce a precise coordination number on a metal center. The d chirality does not alter the native fold, but the side-chain re-orientation modifies the sterics of the metal binding pocket. l-Cys side chains within the coiled-coil structure have previously been shown to rotate substantially from their preferred positions in the apo structure to create a binding site for a tetra-coordinate metal ion. However, here we show by X-ray crystallography that d-Cys side chains are preorganized within a suitable geometry to bind such a ligand. This is confirmed by comparison of the structure of ZnII Cl(CSL16D C)32- to the published structure of ZnII (H2 O)(GRAND-CSL12AL16L C)3- . Moreover, spectroscopic analysis indicates that the CdII geometry observed by using l-Cys ligands (a mixture of three- and four-coordinate CdII ) is altered to a single four-coordinate species when d-Cys is present. This work opens a new avenue for the control of the metal site environment in man-made proteins, by simply altering the binding ligand with its mirror-imaged d configuration. Thus, the use of non-coded amino acids in the coordination sphere of a metal promises to be a powerful tool for controlling the properties of future metalloproteins.

Recent studies have suggested that gut symbionts modulate insect development and reproduction. However, the mechanisms by which gut symbionts modulate host physiologies and the molecules involved in these changes are unclear. To address these questions, we prepared three different groups of the insect Riptortus pedestris: Burkholderia gut symbiont-colonized (Sym) insects, Burkholderia-non-colonized (Apo) insects, and Burkholderia-depleted (Sym(Burk-)) insects, which were fed tetracycline. When the hemolymph proteins of three insects were analyzed by SDS-PAGE, the hexamerin-α, hexamerin-β and vitellogenin-1 proteins of Sym-adults were highly expressed compared to those of Apo- and Sym(Burk-)-insects. To investigate the expression patterns of these three genes during insect development, we measured the transcriptional levels of these genes. The hexamerin-β gene was specifically expressed at all nymphal stages, and its expression was detected 4-5 days earlier in Sym-insect nymphs than that in Apo- and Sym(Burk-)-insects. However, the hexamerin-α and vitellogenin-1 genes were only expressed in adult females, and they were also detected 6-7 days earlier and were 2-fold higher in Sym-adult females than those in the other insects. Depletion of hexamerin-β by RNA interference in 2nd instar Sym-nymphs delayed adult emergence, whereas hexamerin-α and vitellogenin-1 RNA interference in 5th instar nymphs caused loss of color of the eggs of Sym-insects. These results demonstrate that the Burkholderia gut symbiont modulates host development and egg production by regulating production of these three hemolymph storage proteins.

Tocopherols and tocotrienols (usually summed up as vitamin E) are a class of structurally related natural antioxidants. Commonly, only some of the eight classic representatives (four tocopherols and four tocotrienols) are found with varied composition in food. In this study we fractionated 230mg oil from commercial vitamin E supplement capsules by countercurrent chromatography (CCC) and subsequent analysis by gas chromatography with mass spectrometry (GC/MS) of silylated CCC fractions showed that these eight isomers represented only about 70% of total tocopherol compounds. Detailed analysis enabled the detection of 161T3 isomers (α-, γ- and δ-T3) along with 18 tetra- and several penta-unsaturated isomers (tocools), two tocomonoenol isomers, and several degradation products with shorter isoprenoid side chain (apo-tocools). Altogether, over 170 tocool compounds, most likely artefacts which originated from an inappropriate oil refining process were described in this study. Silver ion high performance liquid chromatography (Ag(+)-HPLC) was used to separate one fraction rich in γ-T3 into four peaks each consisting of at least five peaks according to GC/MS. About ten γ-T3 isomers were also detected in rice bran oils from one producer bought retail in Germany.

Clinical resistance to the second-generation antiandrogen enzalutamide in castration-resistant prostate cancer (CRPC), despite persistent androgen receptor (AR) activity in tumors, highlights an unmet medical need for next-generation antagonists. We have identified and characterized tetra-aryl cyclobutanes (CBs) as a new class of competitive AR antagonists that exhibit a unique mechanism of action. These CBs are structurally distinct from current antiandrogens (hydroxyflutamide, bicalutamide, and enzalutamide) and inhibit AR-mediated gene expression, cell proliferation, and tumor growth in several models of CRPC. Conformational profiling revealed that CBs stabilize an AR conformation resembling an unliganded receptor. Using a variety of techniques, it was determined that the AR-CB complex was not recruited to AR-regulated promoters and, like apo AR, remains sequestered in the cytoplasm, bound to heat shock proteins. Thus, we have identified third-generation AR antagonists whose unique mechanism of action suggests that they may have therapeutic potential in CRPC.