Creating Order in Ultrastable Phosphonate Metal-Organic Frameworks via Isolable Hydrogen-Bonded Intermediates.

The stability presented by trivalent metal-organic frameworks (MOFs) makes them an attractive class of materials. With phosphonate-based ligands, crystallization is a challenge, as there are significantly more binding motifs that can be adopted due to the extra oxygen tether compared to carboxylate counterparts and the self-assembly processes are less reversible. Despite this, we have reported charge-assisted hydrogen-bonded metal-organic frameworks (HMOFs) consisting of [Cr(H2O)6]3+ and phosphonate ligands, which were crystallographically characterized. We sought to use these HMOFs as a crystalline intermediate to synthesize ordered Cr(III)-phosphonate MOFs. This can be done by dehydrating the HMOF to remove the aquo ligands around the Cr(III) center, forcing metal-phosphonate coordination. Herein, a new porous HMOF, H-CALF-50, is synthesized and then dehydrated to yield the MOF CALF-50. CALF-50 is ordered, although it is not single crystalline. It does, however, have exceptional stability, maintaining crystallinity and surface area after boiling in water for 3 weeks and soaking in 14.5 M H3PO4 for 24 h and 9 M HCl for 72 h. Computational methods are used to study the HMOF to MOF transformation and give insight into the nature of the structure and the degree of heterogeneity.

The normal appearance of CT myelograms.

PURPOSE: CT myelography has been used since 1976 to diagnose neural compression in the axial skeleton. With the advent of routine MRI, its role in accurately diagnosing neural compression has been questioned as its normal appearances are not defined in the study. In this study, we examine a series of CT myelograms to define the normal appearances of the neural elements of the spine. METHODS: The CT myelograms of patients with unilateral symptoms were examined by four independent physicians. The lateral extent of contrast was examined and recorded. Concordance between the recorded extents was assessed using kappa scores. RESULTS: Thirty-six scans were reviewed. Kappa analysis shows that there is a fair agreement in the lateral extent of contrast at L1, L3 and L4. At L2 and L5, agreement is slight. CONCLUSION: The interpretation of CT myelography shows significant interobserver variability. As a result, the usefulness of this diagnostic tool can be questioned, and if misinterpreted, it could lead to questionable diagnoses and inadvertently erroneous management if used in isolation. These slides can be retrieved under Electronic Supplementary Material.

Activation of Latent Dihydroorotase from Aquifex aeolicus by Pressure.

Elevated hydrostatic pressure was used to probe conformational changes of Aquifex aeolicus dihydroorotase (DHO), which catalyzes the third step in de novo pyrimidine biosynthesis. The isolated protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC). X-ray crystallographic studies of the isolated DHO and of the complex showed that association induces several major conformational changes in the DHO structure. In the isolated DHO, a flexible loop occludes the active site blocking the access of substrates. The loop is mostly disordered but is tethered to the active site region by several electrostatic and hydrogen bonds. This loop becomes ordered and is displaced from the active site upon formation of DHO-ATC complex. The application of pressure to the complex causes its time-dependent dissociation and the loss of both DHO and ATC activities. Pressure induced irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated. However, moderate hydrostatic pressure applied to the isolated DHO subunit mimics the complex formation and reversibly activates the isolated subunit in the absence of ATC, suggesting that the loop has been displaced from the active site. This effect of pressure is explained by the negative volume change associated with the disruption of ionic interactions and exposure of ionized amino acids to the solvent (electrostriction). The interpretation that the loop is relocated by pressure was validated by site-directed mutagenesis and by inhibition by small peptides that mimic the loop residues.

The impact of arterial pedal arch quality and angiosome revascularization on foot tissue loss healing and infrapopliteal bypass outcome.

OBJECTIVE: This study evaluated the effect of pedal arch quality on the amputation-free survival and patency rates of distal bypass grafts and its direct impact on the rate of healing and time to healing of tissue loss after direct angiosome revascularization in patients with critical limb ischemia (CLI). METHODS: Between 2004 and 2011, patients undergoing distal bypass for CLI (Rutherford 4-6) were divided in groups taking into consideration the state of the pedal arch and direct angiosome revascularization (DAR) and non-DAR. Angiography was used to divide the pedal arch into three groups: complete pedal arch (CPA), incomplete pedal arch (IPA), and no pedal arch (NPA). The primary end points were patency rates at 12 months, amputation-free survival at 48 months, and the rate of healing and time to healing of foot tissue loss. RESULTS: A total of 154 patients (75% men) with CLI underwent 167 infrapopliteal bypasses. Patients were a median age of 75 years (range, 46-96 years). Diabetic mellitus was present in 76%, chronic renal failure in 28%, and ischemic heart disease in 44%. The primary patency rates at 1 year in the CPA, IPA, and NPA groups were 58.4%, 54.6%, and 63.8%, respectively (P = .5168), the secondary patency rates were 86.0%, 84.7%, and 88.8%, respectively (P = .8940), and the amputation-free survival at 48 months was 67.2%, 69.7%, and 45.9%, respectively (P = .3883). Tissue loss was present in 141 of the 167 bypasses. In the CPA group, 83% of tissue loss with DAR healed compared with 92% in the non-DAR (median time to healing, 66 vs 74 days). Similarly in the IPA group, 90% with DAR healed compared with 81% in the non-DAR (median time to healing, 96 vs 86 days). In the NPA group, only 75% with DAR healed compared with 73% in the non-DAR (median time to healing, 90 vs 135 days). There was a significant difference in healing and time to healing between the CPA/IPA and NPA groups (P = .0264). CONCLUSIONS: The quality of the pedal arch did not influence the patency or the amputation-free survival rates. However, the rates for healing and time to healing were directly influenced by the quality of the pedal arch rather than the angiosome revascularized.

The mononuclear metal center of type-I dihydroorotase from Aquifex aeolicus.

BACKGROUND: Dihydroorotase (DHO) is a zinc metalloenzyme, although the number of active site zinc ions has been controversial. E. coli DHO was initially thought to have a mononuclear metal center, but the subsequent X-ray structure clearly showed two zinc ions, α and ß, at the catalytic site. Aquifex aeolicus DHO, is a dodecamer comprised of six DHO and six aspartate transcarbamoylase (ATC) subunits. The isolated DHO monomer, which lacks catalytic activity, has an intact α-site and conserved ß-site ligands, but the geometry of the second metal binding site is completely disrupted. However, the putative ß-site is restored when the complex with ATC is formed and DHO activity is regained. Nevertheless, the X-ray structure of the complex revealed a single zinc ion at the active site. The structure of DHO from the pathogenic organism, S. aureus showed that it also has a single active site metal ion. RESULTS: Zinc analysis showed that the enzyme has one zinc/DHO subunit and the addition of excess metal ion did not stimulate catalytic activity, nor alter the kinetic parameters. The metal free apoenzyme was inactive, but the full activity was restored upon the addition of one equivalent of Zn2+ or Co2+. Moreover, deletion of the ß-site by replacing the His180 and His232 with alanine had no effect on catalysis in the presence or absence of excess zinc. The 2.2 Å structure of the double mutant confirmed that the ß-site was eliminated but that the active site remained otherwise intact. CONCLUSIONS: Thus, kinetically competent A. aeolicus DHO has a mononuclear metal center. In contrast, elimination of the putative second metal binding site in amidohydrolyases with a binuclear metal center, resulted in the abolition of catalytic activity. The number of active site metal ions may be a consideration in the design of inhibitors that selectively target either the mononuclear or binuclear enzymes.

FAM129B/MINERVA, a novel adherens junction-associated protein, suppresses apoptosis in HeLa cells.

A recent proteomics study identified FAM129B or MINERVA as a target of the MAP kinase (Erk1/2) signaling cascade in human melanoma cells. Phosphorylation of the protein was found to promote cell invasion and the dissociation of the protein from the cell-cell junctions. Suppression of apoptosis during metastasis is a prerequisite for the survival and spread of cancer cells. During apoptosis, the adherens junctions are disassembled as the dying cell retracts, and new contacts are formed between normal neighboring cells. In this study, we show that FAM129B was cytosolic in exponentially growing HeLa cells but was translocated to the adherens junctions where it colocalized with ß-catenin whenever contact between two or more cells was established. Silencing the FAM129B gene expression by specific siRNAs did not induce apoptosis or inhibit the growth of HeLa cells. However, when apoptosis was induced by exposure to TNFα/cycloheximide or other apoptotic signaling molecules, the onset of apoptosis was accelerated 3-4-fold when FAM129B was depleted. Annexin V binding, the inactivation of the DNA repair enzyme, poly(ADP-ribose) polymerase, and the activation of the caspases occurred more rapidly in the cells lacking FAM129B. The rapid induction of apoptosis in FAM129B knockdown cells was reversed by co-transfection with recombinant FAM129B, indicating that its effect on apoptosis was specific. As apoptosis proceeded, FAM129B was degraded and disappeared from the plasma membrane. Thus, one crucial facet of the mechanism by which FAM129B promotes cancer cell invasion is likely to be the suppression of apoptosis.

Electronic properties of metal-arene functionalized graphene.

We have employed first-principles density-functional calculations to study the electronic characteristics of covalently functionalized graphene by metal-bis-arene chemistry. It is shown that functionalization with M-bis-arene (M = Ti, V, Cr, Mn, Fe) molecules leads to an opening in the bandgap of graphene (up to 0.81 eV for the Cr derivative), and as a result, transforms it from a semimetal to a semiconductor. The bandgap induced by attachment of a metal atom topped by a benzene ring is attributed to modification of π-conjugation and depends on the concentration of functionalizing molecules. This approach offers a means of tailoring the band structure of graphene and potentially its applications for future electronic devices.

Electrical detection of the temperature induced melting transition of a DNA hairpin covalently attached to gold interdigitated microelectrodes.

The temperature induced melting transition of a self-complementary DNA strand covalently attached at the 5' end to the surface of a gold interdigitated microelectrode (GIME) was monitored in a novel, label-free, manner. The structural state of the hairpin was assessed by measuring four different electronic properties of the GIME (capacitance, impedance, dissipation factor and phase angle) as a function of temperature from 25 degrees C to 80 degrees C. Consistent changes in all four electronic properties of the GIME were observed over this temperature range, and attributed to the transition of the attached single-stranded DNA (ssDNA) from an intramolecular, folded hairpin structure to a melted ssDNA. The melting curve of the self-complementary single strand was also measured in solution using differential scanning calorimetry (DSC) and UV absorbance spectroscopy. Temperature dependent electronic measurements on the surface and absorbance versus temperature values measured in solution experiments were analyzed assuming a two-state process. The model analysis provided estimates of the thermodynamic transition parameters of the hairpin on the surface. Two-state analyses of optical melting data and DSC measurements provided evaluations of the thermodynamic transition parameters of the hairpin in solution. Comparison of surface and solution measurements provided quantitative evaluation of the effect of the surface on the thermodynamics of the melting transition of the DNA hairpin.

Characterization and assembly of the Pseudomonas aeruginosa aspartate transcarbamoylase-pseudo dihydroorotase complex.

Pseudomonas aeruginosa is a virulent pathogen that has become more threatening with the emergence of multidrug resistance. The aspartate transcarbamoylase (ATCase) of this organism is a dodecamer comprised of six 37 kDa catalytic chains and six 45 kDa chains homologous to dihydroorotase (pDHO). The pDHO chain is inactive but is necessary for ATCase activity. A stoichiometric mixture of the subunits associates into a dodecamer with full ATCase activity. Unlike other known ATCases, the P. aeruginosa catalytic chain does not spontaneously assemble into a trimer. Chemical-crosslinking and size-exclusion chromatography showed that P. aeruginosa ATCase is monomeric which accounts for its lack of catalytic activity since the active site is a composite comprised of residues from adjacent monomers in the trimer. Circular dichroism spectroscopy indicated that the ATCase chain adopts a structure that contains secondary structure elements although neither the ATCase nor the pDHO subunits are very stable as determined by a thermal shift assay. Formation of the complex increases the melting temperature by about 30°C. The ATCase is strongly inhibited by all nucleotide di- and triphosphates and exhibits extreme cooperativity. Previous studies suggested that the regulatory site is located in an 11-residue extension of the amino end of the catalytic chain. However, deletion of the extensions did not affect catalytic activity, nucleotide inhibition or the assembly of the dodecamer. Nucleotides destabilized the dodecamer which probably accounts for the inhibition and apparent cooperativity of the substrate saturation curves. Contrary to previous interpretations, these results suggest that P. aeruginosa ATCase is not allosterically regulated by nucleotides.

Dihydroorotase from the hyperthermophile Aquifex aeolicus is activated by stoichiometric association with aspartate transcarbamoylase and forms a one-pot reactor for pyrimidine biosynthesis.

In prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPS), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), are commonly expressed separately and either function independently (Escherichia coli) or associate into multifunctional complexes (Aquifex aeolicus). In mammals the enzymes are expressed as a single polypeptide chain (CAD) in the order CPS-DHO-ATC and associate into a hexamer. This study presents the three-dimensional structure of the noncovalent hexamer of DHO and ATC from the hyperthermophile A. aeolicus at 2.3 A resolution. It is the first structure of any multienzyme complex in pyrimidine biosynthesis and is a possible model for the core of mammalian CAD. The structure has citrate, a near isosteric analogue of carbamoyl aspartate, bound to the active sites of both enzymes. Three active site loops that are intrinsically disordered in the free, inactive DHO are ordered in the complex. The reorganization also changes the peptide bond between Asp153, a ligand of the single zinc atom in DHO, and Gly154, to the rare cis conformation. In the crystal structure, six DHO and six ATC chains form a hollow dodecamer, in which the 12 active sites face an internal reaction chamber that is approximately 60 A in diameter and connected to the cytosol by narrow tunnels. The entrances and the interior of the chamber are both electropositive, which suggests that the architecture of this nanoreactor modifies the kinetics of the bisynthase, not only by steric channeling but also by preferential escape of the product, dihydroorotase, which is less negatively charged than its precursors, carbamoyl phosphate, aspartate, or carbamoyl aspartate.

Pressure-induced activation of latent dihydroorotase from Aquifex aeolicus as revealed by high pressure protein crystallography.

Dihydroorotase (DHOase) is involved in the de novo synthesis of pyrimidine in virtually all organisms, and it is usually associated with two other enzymes found in this biosynthetic pathway, carbamylphosphate synthetase and/or aspartate transcarbamylase (ATCase). In the hyperthermophilic bacterium Aquifex aeolicus, ATCase and DHOase are noncovalently associated. Upon dissociation, ATCase keeps its activity entirely while DHOase is totally inactivated. It was previously shown that high pressure fully restores the activity of this isolated DHOase. On the basis of kinetic studies, site-directed mutagenesis and the use of peptides mimicking loop A, a loop that appears to block access to the active site, was proposed that this pressure-induced reactivation was due to the decrease in the volume of the system, -ΔV, resulting from the disruption of known ionic interactions between the loop and the main part of the protein. In this study, this interpretation is more precisely demonstrated by the determination of the crystallographic structure of isolated DHOase under pressure. In addition to the loop displacements, pressure induces a discrete rearrangement of the catalytic site aspartate 305, an effect that might additionally contribute to the reactivation of this enzyme.

Purification of an Fc-fusion biologic: clearance of multiple product related impurities by hydrophobic interaction chromatography.

An hydrophobic interaction chromatography step was developed for the large-scale production of an Fc-fusion biologic. Two abundant product-related impurities were separated from the active monomer using a Butyl resin and a simple step-wash and step-elution strategy. Capacity and resolution of the HIC step was optimal when sodium sulfate was employed as the lyotropic salt and pore size of the Butyl resin was 750A. Factorial analysis identified critical parameters for the Butyl chromatography and an operating window capable of delivering high product quality and yield over a broad column loading range.

Protein kinase C modulates the up-regulation of the pyrimidine biosynthetic complex, CAD, by MAP kinase.

The multifunctional protein CAD initiates de novo pyrimidine biosynthesis in mammalian cells. CAD is activated by MAP kinase (Erk1/2) just prior to the S phase of the cell cycle, when the demand for pyrimidine nucleotides is greatest, and down-regulated as the cells emerge from S phase by protein kinase A (PKA) phosphorylation. MAP kinase phosphorylates Thr456, while PKA phosphorylates Ser1406 and Ser1859, although only Ser1406 is involved in regulation. LC/mass spectrometry showed that Ser1873, a residue that lies within a putative protein kinase C (PKC) consensus sequence is also phosphorylated. Purified CAD was reacted with ATP and a panel of eight PKC isozymes. Most isozymes resulted in limited CAD phosphorylation, but the delta and epsilon isozymes were most effective. While the level of Thr456 phosphorylation is very low in confluent cells, exposure of stationary BHK 165-23 cells to the PKC activator, phorbol 12-myristate-13-acetate (PMA) resulted in a 3-fold increase in the modification of this residue. The stimulation of Thr456 phosphorylation was blocked by PKC inhibitors. The PKA inhibitor, H-89, also stimulated PMA-induced Thr456 modification probably because PKA mediated phosphorylation of CAD Ser1406 antagonizes the MAP kinase phosphorylation. Thus, the extent of Thr456 phosphorylation and the activation of pyrimidine biosynthesis depend on the synergistic and antagonistic interactions of three signaling pathways, MAP kinase, PKC and PKA. Deletions mutants lacking the putative PKC site, Ser1873 do not exhibit PMA induced Thr456 phosphorylation. We conclude that the activating MAP kinase phosphorylation of CAD proceeds through a PKC dependent pathway.

The crystal structure of a novel, latent dihydroorotase from Aquifex aeolicus at 1.7A resolution.

Dihydroorotases (EC 3.5.2.3) catalyze the reversible cyclization of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The X-ray structures of Aquifex aeolicus dihydroorotase in two space groups, C222(1) and C2, were determined at a resolution of 1.7A. These are the first structures of a type I dihydroorotase, a class of molecules that includes the dihydroorotase domain of mammalian CAD. The type I enzymes are more ancient and larger, at 45 kDa, than the type II enzymes exemplified by the 38 kDa Escherichia coli dihydroorotase. Both dihydroorotases are members of the metallo-dependent hydrolase superfamily, whose members have a distorted "TIM barrel" domain containing the active site. However, A.aeolicus dihydroorotase has a second, composite domain, which the E.coli enzyme lacks and has only one of the two zinc atoms present in the E.coli enzyme. A.aeolicus dihydroorotase is unique in exhibiting significant activity only when complexed with aspartate transcarbamoylase, whereas the E.coli dihydroorotase and the CAD dihydroorotase domain are active as free proteins. The latency of A.aeolicus dihydroorotase can be related to two differences between its structure and that of E.coli dihydroorotase: (1) the monoclinic structure has a novel cysteine ligand to the zinc that blocks the active site and possibly functions as a "cysteine switch"; and (2) active site residues that bind the substrate in E.coli dihydroorotase are located in disordered loops in both crystal structures of A.aeolicus dihydroorotase and may function as a disorder-to-order "entropy switch".

Cell-based assays for identification of novel double-strand break-inducing agents.

BACKGROUND: We are developing cell-based assays to identify anticancer agents that are selectively toxic to cells with defined mutations. As a test, we used a three-stage strategy to screen compounds from the National Cancer Institute's repository for agents that are selectively toxic to double-strand break repair-deficient yeast cells. METHODS: Compounds identified in the screen were further analyzed by use of yeast and vertebrate cell-based and in vitroassays to distinguish between topoisomerase I and II poisons. RESULTS: Of the more than 85 000 compounds screened, 126 were selectively toxic to yeast deficient in DNA double-strand break repair. Eighty-seven of these 126 compounds were structurally related to known topoisomerase poisons, and 39 were not. Twenty-eight of the 39 were characterized, and we present data for eight of the compounds. Among these eight compounds, we identified two novel topoisomerase II poisons (NSC 327929 and NSC 638432) that were equipotent to etoposide in biochemical tests and in cells, five (NSC 63599, NSC 65601, NSC 380271, NSC 651646, and NSC 668370) with topoisomerase I-dependent toxicity in yeast that induced DNA damage and toxicity in mammalian cells, and one (NSC 610898) that directly bound to DNA and induced strand breaks. CONCLUSIONS: Cell-based assays can be used to identify molecules that are selectively toxic to cells with a predetermined genetic background, including mutations in genes involved in the cell cycle and its checkpoints, for which there are currently no selectively toxic compounds.

Antimony/Graphitic Carbon Composite Anode for High-Performance Sodium-Ion Batteries.

Although the room-temperature rechargeable sodium-ion battery has emerged as an attractive alternative energy storage solution for large-scale deployment, major challenges toward practical sodium-ion battery technology remain including identification and engineering of anode materials that are both technologically feasible and economical. Herein, an antimony-based anode is developed by incorporating antimony into graphitic carbon matrices using low-cost materials and scalable processes. The composite anode exhibits excellent overall performance in terms of packing density, fast charge/discharge capability and cyclability, which is enabled by the conductive and compact graphitic network. A full cell design featuring this composite anode with a hexacyanometallate cathode achieves superior power output and low polarization, which offers the potential for realizing a high-performance, cost-effective sodium-ion battery.

Direct demonstration of carbamoyl phosphate formation on the C-terminal domain of carbamoyl phosphate synthetase.

Carbamoyl phosphate synthetase synchronizes the utilization of two ATP molecules at duplicated ATP-grasp folds to catalyze carbamoyl phosphate formation. To define the dedicated functional role played by each of the two ATP sites, we have carried out pulse/labeling studies using the synthetases from Aquifex aeolicus and Methanococcus jannaschii, hyperthermophilic organisms that encode the two ATP-grasp folds on separate subunits. These studies allowed us to differentially label each active site with [gamma-(32)P]ATP and determine the fate of the labeled gamma-phosphate in the synthetase reaction. Our results provide the first direct demonstration that enzyme-catalyzed transfer of phosphate from ATP to carbamate occurs on the more C-terminal of the two ATP-grasp folds. These findings rule out one mechanism proposed for carbamoyl phosphate synthetase, where one ATP acts as a molecular switch, and provide additional support for a sequential reaction mechanism where the gamma-phosphate groups of both ATP molecules are transferred to reactants. CP synthesis by subunit C in our single turnover pulse/chase assays did not require subunit N, but subunit N was required for detectable CP synthesis in the traditional continuous assay. These findings suggest that cross-talk between domain N and C is required for product release from subunit C.

"One plate/three-reporter" assay format for the detection and validation of yeast two-hybrid interactions.

We describe a novel assay format for the Gal4-based yeast two-hybrid-system, in which the readout from three different reporter genes is measured sequentially in a single microplate. Activation of the URA3, MEL1, and lacZ reporters in response to a protein-protein interaction is monitored by measuring sequentially: (i) growth in medium lacking uracil, (ii) alpha-galactosidase activity, and (iii) beta-galactosidase. The data thus generated permit elimination of many false positive signals and provide a preliminary measurement of reporter activation-strength that may be confirmed by further analysis. The assay procedure is inexpensive and requires few liquid-handling steps. It is appropriate for automated high-throughput interaction mating assays, validation of putative interactor strains and hybrid-protein self-activator tests.

The diabetic foot: the importance of coordinated care.

Because of the severe morbidity and mortality associated with diabetes, diabetic foot care is an essential component of a peripheral vascular service. The goal of this article is to describe the vascular diabetic foot care pathway and how the coordinated foot care service for diabetic patients is delivered at King's College Hospital, London.