Exploring Inner-Sphere Water Interactions of Fe(II) and Co(II) Complexes of 12-Membered Macrocycles To Develop CEST MRI Probes.

Several paramagnetic Co(II) and Fe(II) macrocyclic complexes were prepared with the goal of introducing a bound water ligand to produce paramagnetically shifted water 1H resonances and for paramagnetic chemical exchange saturation transfer (paraCEST) applications. Three 12-membered macrocycles with amide pendent groups including 1,7-bis(carbamoylmethyl)-1,4,7,10-tetraazacyclodocane (DCMC), 4,7,10-tris(carbamoylmethyl)-,4,7,10-triaza-12-crown-ether (N3OA), and 4,10-bis(carbamoylmethyl)-4,10-diaza-12-crown-ether (NODA) were prepared and their Co(II) complexes were characterized in the solid state and in solution. The crystal structure of [Co(DCMC)]Br2 featured a six-coordinated Co(II) center with distorted octahedral geometry, while [Co(NODA)(OH2)]Cl2 and [Co(N3OA)](NO3)2 were seven-coordinated. The analogous Fe(II) complexes of NODA and NO3A were successfully prepared, but the complex of DCMC oxidized rapidly to the Fe(III) form. Similarly, [Fe(NODA)]2+ oxidized over several days, forming crystals of the Fe(III) complex isolated as the µ-O bridged dimer. Magnetic susceptibility values and paramagnetic NMR spectra of the Fe(II) complexes of NODA and N3OA, as well as Co(II) complexes of DCMC, NODA, and N3OA, were consistent with high spin complexes. CEST peaks ranging from 60 ppm to 70 ppm, attributed to NH groups of the amide pendents, were identified. Variable-temperature 17O NMR spectra of Co(II) and Fe(II) NODA complexes were consistent with rapid exchange of the water ligand with bulk water. Notably, the Co(II) and Fe(II) complexes presented here produced substantial paramagnetic shifts of bulk water 1H resonances, independent of having an inner-sphere water.

Saccharomyces jurei sp. nov., isolation and genetic identification of a novel yeast species from Quercus robur.

Two strains, D5088T and D5095, representing a novel yeast species belonging to the genus Saccharomyces were isolated from oak tree bark and surrounding soil located at an altitude of 1000 m above sea level in Saint Auban, France. Sequence analyses of the internal transcribed spacer (ITS) region and 26S rRNA D1/D2 domains indicated that the two strains were most closely related to Saccharomyces mikatae and Saccharomyces paradoxus. Genetic hybridization analyses showed that both strains are reproductively isolated from all other Saccharomyces species and, therefore, represent a distinct biological species. The species name Saccharomyces jurei sp. nov. is proposed to accommodate these two strains, with D5088T (=CBS 14759T=NCYC 3947T) designated as the type strain.

Apiotrichum terrigenum sp. nov., a soil-associated yeast found in both the UK and mainland Europe.

Five arthroconidium-producing yeast strains representing a novel Trichosporon-like species were independently isolated from the UK, Hungary and Norway. Two strains (Bio4T and Bio21) were isolated from biogas reactors used for processing grass silage, with a third strain (S8) was isolated from soil collected at the same UK site. Two additional strains were isolated in mainland Europe, one from soil in Norway (NCAIM Y.02175) and the other from sewage in Hungary (NCAIM Y.02176). Sequence analyses of the D1/D2 domains of the LSU rRNA gene and internal transcribed spacer (ITS) region indicated that the novel species belongs to the recently reinstated genus Apiotrichum and is most closely related to Apiotrichum scarabaeorum, a beetle-associated species first found in South Africa. Despite having similar physiological characteristics, the two species can be readily distinguished from one another by ITS sequencing. The species name Apiotrichum terrigenum sp. nov. is proposed to accommodate these strains, with Bio4T (=CBS 11373T=NCYC 3540T) designated as the type strain. The Mycobank deposit number is MB817431.

Six, Seven or Eight Coordinate Fe(II) , Co(II) or Ni(II) Complexes of Amide-Appended Tetraazamacrocycles for ParaCEST Thermometry.

Fe(II) , Co(II) and Ni(II) complexes of two tetraazamacrocycles (1,4,8,11-tetrakis(carbamoylmethyl)-1,4,8,11-tetraazacyclotetradecane (L1) and 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (L2) show promise as paraCEST agents for registration of temperature (paraCEST=paramagnetic chemical exchange saturation transfer). The Fe(II) , Co(II) and Ni(II) complexes of L1 show up to four CEST peaks shifted ≤112 ppm, whereas analogous complexes of L2 show only a single CEST peak at ≤69 ppm. Comparison of the temperature coefficients (CT ) of the CEST peaks of [Co(L2)](2+) , [Fe(L2)](2+) , [Ni(L1)](2+) and [Co(L1)](2+) showed that a CEST peak of [Co(L1)](2+) gave the largest CT (-0.66 ppm (o) C(-1) at 4.7 T). NMR spectral and CEST properties of these complexes correspond to coordination complex symmetry as shown by structural data. The [Ni(L1)](2+) and [Co(L1)](2+) complexes have a six-coordinate metal ion bound to the 1-, 4-amide oxygen atoms and four nitrogen atoms of the tetraazamacrocycle. The [Fe(L2)](2+) complex has an unusual eight-coordinate Fe(II) bound to four amide oxygen atoms and four macrocyclic nitrogen atoms. For [Co(L2)](2+) , one structure has seven-coordinate Co(II) with three bound amide pendents and a second structure has a six-coordinate Co(II) with two bound amide pendents.

Kazachstania yasuniensis sp. nov., an ascomycetous yeast species found in mainland Ecuador and on the Galápagos.

Seven strains representing a novel yeast species belonging to the genus Kazachstania were found at several collection sites on both mainland Ecuador (Yasuní National Park) and the Galápagos (Santa Cruz Island). Two strains (CLQCA 20-132(T) and CLQCA 24SC-045) were isolated from rotten wood samples, two further strains (CLQCA 20-280 and CLQCA 20-348) were isolated from soil samples, and three strains (CLQCA 20-198, CLQCA 20-374 and CLQCA 20-431) were isolated from decaying fruits. Sequence analyses of the D1/D2 domains of the LSU rRNA gene and ribosomal internal transcribed spacer (ITS) region indicated that the novel species is most closely related to Kazachstania servazzii and Kazachstania unispora. Although the strains could not be distinguished from one another based upon their differing geographical origins, they could be differentiated according to their isolation source (fruit, soil or wood) by ITS sequencing. The species name Kazachstania yasuniensis sp. nov. is proposed to accommodate these strains, with CLQCA 20-132(T) ( = CBS 13946(T) = NCYC 4008(T)) designated the type strain.

Wnt pathway inhibition via the targeting of Frizzled receptors results in decreased growth and tumorigenicity of human tumors.

The Wnt/ß-catenin pathway, which signals through the Frizzled (Fzd) receptor family and several coreceptors, has long been implicated in cancer. Here we demonstrate a therapeutic approach to targeting the Wnt pathway with a monoclonal antibody, OMP-18R5. This antibody, initially identified by binding to Frizzled 7, interacts with five Fzd receptors through a conserved epitope within the extracellular domain and blocks canonical Wnt signaling induced by multiple Wnt family members. In xenograft studies with minimally passaged human tumors, this antibody inhibits the growth of a range of tumor types, reduces tumor-initiating cell frequency, and exhibits synergistic activity with standard-of-care chemotherapeutic agents.

Wickerhamomyces arborarius f.a., sp. nov., an ascomycetous yeast species found in arboreal habitats on three different continents.

Five strains representing a novel yeast species belonging to the genus Wickerhamomyces were independently isolated from Ecuador, Taiwan and the USA. One strain (CLQCA 10-161(T)) was isolated from the white flower of an unidentified plant species collected in the Maquipucuna cloud forest reserve, near Quito, in Ecuador. A second strain (GY7L12) was isolated from the leaf of a Chinese sumac or nutgall tree (Rhus chinensis 'roxburghiana') collected in the Taoyuan mountain area, Kachsiung, in Taiwan. Three additional strains (A543, A546 and A563) were isolated from two species of wood-boring beetle (Xyleborus glabratus and Xyleborinus saxeseni) collected near Clyo, Georgia, USA. Analysis of the D1/D2 domains of the LSU rRNA gene indicated that the novel species belongs to the genus Wickerhamomyces, and is most closely related to Wickerhamomyces sydowiorum, an insect-associated species predominantly found in South Africa. The North American and Taiwanese strains have identical internal transcribed spacer (ITS) sequences and can be distinguished from the Ecuadorian strain based on a single nucleotide substitution in the ITS1 region. The species name of Wickerhamomyces arborarius f.a., sp. nov. is proposed to accommodate these strains, with CLQCA 10-161(T) ( = CBS 12941(T) = NCYC 3743(T)) designated the type strain.

Candida ecuadorensis sp. nov., an ascomycetous yeast species found in two separate regions of Ecuador.

In the course of an on-going study aimed at cataloguing the natural yeast biodiversity found in Ecuador, two strains (CLQCA 13-025 and CLQCA 20-004(T)) were isolated from samples of cow manure and rotten wood collected in two separate provinces of the country (Orellana and Bolívar). These strains were found to represent a novel yeast species based on the sequences of their D1/D2 domain of the large-subunit (LSU) rRNA gene and their physiological characteristics. Phylogenetic analysis based on LSU D1/D2 sequences revealed this novel species to belong to the Metschnikowia clade and to be most closely related to Candida suratensis, a species recently discovered in a mangrove forest in Thailand. The species name of Candida ecuadorensis sp. nov. is proposed to accommodate these strains, with strain CLQCA 20-004(T) (=CBS 12653(T) = NCYC 3782(T)) designated as the type strain.

2-(Pyridin-4-yl)-1H-benzimidazole.

The title compound, C12H9N3, is an unhydrated analogue of the previously reported trihydrate. The mol-ecule is essentially planar, with a 3.62 (11)° angle between the pyridine and benzimidazole planes. In the crystal, N-H⋯N hydrogen bonds result in chains of mol-ecules parallel to [010], which are additionally linked by weak π-π stacking inter-actions [centroid-centroid distance = 3.7469 (17) Å], resulting in extended sheets of molecules parallel to (103).

Co(II) complexes of tetraazamacrocycles appended with amide or hydroxypropyl groups as paraCEST agents.

Co(II) complexes of 1,4,7,10-tetraazacyclododecane (CYCLEN) or 1,4,8,11-tetraazacyclotetradecane (CYCLAM) with 2-hydroxypropyl or carbamoylmethyl (amide) pendants are studied with the goal of developing paramagnetic chemical exchange saturation transfer (paraCEST) agents. Single-crystal X-ray diffraction studies show that two of the coordination cations with hexadentate ligands, [Co(DHP)]2+ and [Co(BABC)]2+, form six-coordinate complexes; whereas two CYCLEN-based complexes with potentially octadentate ligands, [Co(THP)]2+ and [Co(HPAC)]2+, are seven-coordinate with only three of the four pendant groups bound to the metal center. 1H NMR spectra of these complexes suggest that the six-coordinate complexes are present as a single isomer in aqueous solution. For the complexes which are seven-coordinate in the solid state, one is highly fluxional in aqueous solution on the NMR time scale ([Co(HPAC)]2+), whereas the NMR spectrum of [Co(THP)]2+ is consistent with an eight-coordinate complex with all pendants bound. Co(II) complexes of CYCLEN derivatives show CEST effects of low intensity that are assigned to NH or OH groups of the pendants. One complex, [Co(DHP)]2+, shows a highly-shifted CEST peak at 113 ppm versus bulk water, attributed to OH protons. However, the CEST effect is largest for two Co(II) CYCLAM-based complexes with coordinated amide groups that undergo NH proton exchange. All five complexes are inert towards dissociation in buffered solutions containing carbonate and phosphate and towards trans-metalation by excess Zn(II). These data give insight into the production of an intense CEST effect for tetraazamacrocyclic complexes with pendant groups containing NH or OH exchangeable protons. The intense and highly shifted CEST peak(s) of the CYCLAM-based complexes suggest that they are promising for further development as paraCEST agents.

Saturnispora quitensis sp. nov., a yeast species isolated from the Maquipucuna cloud forest reserve in Ecuador.

A single strain, CLQCA-10-114(T), representing a novel yeast species belonging to the genus Saturnispora was isolated from the fruit of an unidentified species of bramble (Rubus sp.), collected from the Maquipucuna cloud forest reserve, near Quito, in Ecuador. Sequence analyses of the D1/D2 domains of the large-subunit rRNA gene and ribosomal internal transcribed spacer region indicated that the novel species is most closely related to the recently described species Saturnispora gosingensis, isolated from the fruiting body of a mushroom collected in Taiwan, and Saturnispora hagleri, a Drosophila-associated yeast found in Brazil. The name Saturnispora quitensis sp. nov. is proposed to accommodate this strain; the type strain is CLQCA-10-114(T) (=CBS 12184(T)=NCYC 3744(T)).

Candida northwykensis sp. nov., a novel yeast isolated from the gut of the click beetle Melanotus villosus.

Two yeast morphotypes, BET 4(T) and BET 7, were isolated from the gut of click beetle Melanotus villosus. Click beetles were collected from the decaying timber within the woodlands of North Wyke Research, South West England, UK (latitude, 50°46'29″N; longitude, 3°55'23″W). Morphotype BET 7 was identified as Debaryomyces hansenii, and the other morphotype, BET 4(T), was found to differ from Priceomyces castillae and Priceomyces haplophilus, its closest phylogenetic neighbours, by 5.0% with respect to the nucleotide sequence of the D1/D2 domain of the large-subunit (LSU) rRNA gene, and by 8.0% with respect to the ribosomal internal-transcribed spacer (ITS) region. BET 4(T) also differ from P. castillae and P. haplophilus in a number of different phenotypic characteristics. Thus, based on the unique nucleotide sequences of its D1/D2 domain and ITS region, its physiological characteristics and an inability to sporulate, strain BET 4(T) is assigned the status of a new species of Candida, for which the name Candida northwykensis sp. nov., is proposed. The type strain is BET 4(T) (NCYC 3525(T) = CBS 11370(T)).

Cryptococcus shivajii sp. nov.: a novel basidiomycetous yeast isolated from biogas reactor.

Five yeast morphotypes were isolated from biogas reactors at North Wyke Research, Okehampton, UK. Out of the five morphotypes, four were identified as known species. In contrast, the fifth morphotype strain, Bio10(T), was found to differ from Bullera dendrophila and Kwoniella mangroviensis, its closest phylogenetic neighbours, by 2.6-2.9% with respect to the nucleotide sequence of the D1/D2 domain of the 26S rRNA gene and by 5.6-6.2% with respect to the internal transcribed spacer 1 (ITS1)-5.8S rRNA gene-ITS2 region. Bio10(T) also differs from these two species by a number of phenotypic characteristics. Thus, based on the phenotypic differences and phylogenetic analysis, strain Bio10(T) is assigned the status of a new species of Cryptococcus, for which the name Cryptococcus shivajii sp. nov. is proposed. The type strain is Bio10(T) (NCYC 3541(T) = CBS 11374(T)).

Isomeric Co(ii) paraCEST agents as pH responsive MRI probes.

A newly discovered isomer of Co(ii) (1,4,8,11-tetrakis(carbamoylmethyl)-1,4,8,11-tetraazacyclotetradecane = CCRM) produces four highly paramagnetically shifted chemical exchange saturation transfer (CEST) peaks. The 1,8-pendants of the complex are bound in a trans-arrangement to produce a Co(ii) complex of increased kinetic inertness. The isomers have a stabilized Co(ii) center (E1/2 of 540 to 550 mV versus SHE). Both the 1,8 and the 1,4-isomer are excellent pH probes in solution and in tissue homogenate by virtue of their highly paramagnetically shifted amide protons. These isomers produce both a ratiometric pH readout as well as amide proton exchange rate constants that correlate to pH.

Candida carvajalis sp. nov., an ascomycetous yeast species from the Ecuadorian Amazon jungle.

In the course of a yeast biodiversity survey of different ecological habitats found in Ecuador, two yeast strains (CLQCA 20-011(T) and CLQCA20-014) were isolated from samples of rotten wood and fallen leaf debris collected at separate sites in the central region of the Ecuadorian Amazonia. These strains were found to represent a novel yeast species based on the sequences of their D1/D2 domain of the large-subunit (LSU) rRNA gene and their physiological characteristics. Phylogenetic analysis based on LSU D1/D2 sequences revealed this novel species to be most closely related to Candida asparagi, Candida fructus, Candida musae and two as yet undescribed Candida species, with the six yeast taxa collectively forming a distinct species group within the Clavispora clade. The species name of Candida carvajalis sp. nov. is proposed to accommodate these strains, with CLQCA 20-011(T) (NCYC 3509(T), CBS 11361(T)) designated as the type strain.

Diverse Lineages of Candida albicans Live on Old Oaks.

The human pathogen Candida albicans is considered an obligate commensal of animals, yet it is occasionally isolated from trees, shrubs, and grass. We generated genome sequence data for three strains of C. albicans that we isolated from oak trees in an ancient wood pasture, and compared these to the genomes of over 200 clinical strains. C. albicans strains from oak are similar to clinical C. albicans in that they are predominantly diploid and can become homozygous at the mating locus through whole-chromosome loss of heterozygosity. Oak strains differed from clinical strains in showing slightly higher levels of heterozygosity genome-wide. Using phylogenomic analyses and in silico chromosome painting, we show that each oak strain is more closely related to strains from humans and other animals than to strains from other oaks. The high genetic diversity of C. albicans from old oaks shows that they can live in this environment for extended periods of time.

A structure-based database of antibody variable domain diversity.

The diversity of natural antibodies is limited by the genetic mechanisms that engender diversity and the functional requirements of antigen binding. Using an in vitro-evolved autonomous heavy chain variable domain (V(H)H-RIG), we have investigated the limits of structurally-tolerated diversity in the three complementarity-determining regions and a fourth loop within the third framework region. We determined the X-ray crystal structure of the V(H)H-RIG domain at 1.9A resolution and used it to guide the design of phage-displayed libraries encompassing the four loops. The libraries were subjected to selections for structural stability, and a database of structurally-tolerated sequences was compiled from the sequences of approximately 1000 unique clones. The results reveal that all four loops accommodate significantly greater diversity than is observed in nature. Thus, it appears that most sequence biases in the natural immune repertoire arise from factors other than structural constraints and, consequently, it should be possible to enhance the functions of antibodies significantly through in vitro evolution.

Contributions of CDR3 to V H H domain stability and the design of monobody scaffolds for naive antibody libraries.

Camelids produce functional antibodies devoid of light chains. Autonomous heavy chain variable (V(H)H) domains in these molecules have adapted to the absence of the light chain in the following ways: bulky hydrophobic residues replace small aliphatic residues in the former light chain interface, and residues from the third complementarity-determining region (CDR3) pack against the framework and stabilize the global V(H)H domain fold. To determine the specific roles of CDR3 residues in framework stabilization, we used nai;ve phage-displayed libraries, combinatorial alanine-scanning mutagenesis and biophysical characterization of purified proteins. Our results indicate that in the most stable scaffolds, the structural residues in CDR3 reside near the boundaries of the loop and pack against the framework to form a small hydrophobic core. These results allow us to differentiate between structural CDR3 residues that should remain fixed, and CDR3 residues that are tolerant to substitution and can therefore be varied to generate functional diversity within phage-displayed libraries. These methods and insights can be applied to the rapid design of heavy chain scaffolds for the identification of novel ligands using synthetic, antibody-phage libraries. In addition, they shed light on the relationships between CDR3 sequence diversity and the structural stability of the V(H)H domain fold.

Candida theae sp. nov., a new anamorphic beverage-associated member of the Lodderomyces clade.

Four strains representing a novel yeast species belonging to the genus Candida were independently isolated in Taiwan and Ecuador. Two strains (G17(T) and G31) were isolated in Taiwan, by pellet precipitation from plastic-bottled tea drinks produced in Indonesia, while two additional strains (CLQCA 10-049 and CLQCA 10-062) were recovered from ancient chicha fermentation vessels found in tombs in Quito, Ecuador. These four strains were morphologically, and phylogenetically identical to each other. No sexual reproduction was observed on common sporulation media. Large-subunit (LSU) rRNA gene sequence analysis revealed the four strains to belong to the Lodderomyces clade, closely related to members of the Candida parapsilosis species complex. The four strains, which have identical LSU D1/D2 sequences, differ from their closest phylogenetic neighbors, Candida orthopsilosis and Candida parapsilosis, by 6-9 nt substitutions, respectively. Physiologically, the four strains are similar to Candida parapsilosis, although they can be distinguished from their closest relative by the assimilation of arbutin, nitrite, and creatine. The Indonesian and Ecuadorian strain sets can also be distinguished from one another based on ITS sequencing, differing by 4 substitutions in ITS1 and 1 single nucleotide indel in ITS2. Collectively, the results indicate that the four strains represent a previously unrecognized species of Candida. The name Candida theae sp. nov. is proposed to accommodate these strains, with G-17(T) (BCRC 23242(T)=CBS 12239(T)=ATCC MYA-4746(T)) designated as the type strain.

Americas Antibody Congress: September 21-23, 2009, Washington, D.C.

The Americas Antibody Congress, organized by Terrapinn, was held in Washington, D.C. on September 21-23, 2009. The conference sessions were expertly moderated by Herren Wu (MedImmune), and featured 26 speakers who discussed a wide variety of topics relevant to antibody research and development. In addition to the presentations, the conference included four panel sessions during which unscripted dialog between panelist and the audience was encouraged. The topics of the panel sessions were: (1) identifying antibody targets; (2) strategies to assess immunogenicity; (3) antibody drug conjugates; (4) next generation protein types.