Mucosal Immunization of Cynomolgus Macaques with Two Serotypes of Live Poliovirus Vectors Expressing Simian Immunodeficiency Virus Antigens: Stimulation of Humoral, Mucosal, and Cellular Immunity

Poliovirus live virus vectors are a candidate recombinant vaccine system. Previous studies using this system showed that a live poliovirus vector expressing a foreign antigen between the structural and nonstructural proteins generates both antibody and cytotoxic T-lymphocyte responses in mice. Here we describe a novel in vitro method of cloning recombinant polioviruses involving a hybrid-PCR approach. We report the construction of recombinant vectors of two different serotypes of poliovirus-expressing simian immunodeficiency virus (SIV) antigens and the intranasal and intravenous inoculations of four adult cynomolgus macaques with these poliovirus vectors expressing the SIV proteins p17(gag) and gp41(env). All macaques generated a mucosal anti-SIV immunoglobulin A (IgA) response in rectal secretions. Two of the four macaques generated mucosal antibody responses detectable in vaginal lavages. Strong serum IgG responses lasting for at least 1 year were detected in two of the four monkeys. SIV-specific T-cell lymphoproliferative responses were detected in three of the four monkeys. SIV-specific cytotoxic T lymphocytes were detected in two of the four monkeys. This is the first report of poliovirus-elicited vaginal IgA or cytotoxic T lymphocytes in any naturally infectable primate, including humans. These findings support the concept that a live poliovirus vector is a potentially useful delivery system that elicits humoral, mucosal, and cellular immune responses against exogenous antigens.     

Individual heterogeneity in fitness in a long‐lived herbivore

Heterogeneity in the intrinsic quality and nutritional condition of individuals affects reproductive success and consequently fitness. Black brant (Branta bernicla nigricans) are long‐lived, migratory, specialist herbivores. Long migratory pathways and short summer breeding seasons constrain the time and energy available for reproduction, thus magnifying life‐history trade‐offs. These constraints, combined with long lifespans and trade‐offs between current and future reproductive value, provide a model system to examine the role of individual heterogeneity in driving life‐history strategies and individual heterogeneity in fitness. We used hierarchical Bayesian models to examine reproductive trade‐offs, modeling the relationships between within‐year measures of reproductive energy allocation and among‐year demographic rates of individual females breeding on the Yukon‐Kuskokwim Delta, Alaska, using capture–recapture and reproductive data from 1988 to 2014. We generally found that annual survival tended to be buffered against variation in reproductive investment, while breeding probability varied considerably over the range of clutch size‐laying date combinations. We provide evidence for relationships between breeding probability and clutch size, breeding probability and nest initiation date, and an interaction between clutch size and initiation date. Average lifetime clutch size also had a weak positive relationship with apparent survival probability. Our results support the use of demographic buffering strategies for black brant. These results also indirectly suggest associations among environmental conditions during growth, fitness, and energy allocation, highlighting the effects of early growth conditions on individual heterogeneity, and subsequently, lifetime reproductive investment.     

Oxadiazole derivatives as a novel class of antimitotic agents: Synthesis, inhibition of tubulin polymerization, and activity in tumor cell lines

Oxadiazole derivatives were synthesized and evaluated for their ability to inhibit tubulin polymerization and to cause mitotic arrest in tumor cells. The most potent compounds inhibited tubulin polymerization at concentrations below 1 microM. Lead analogs caused mitotic arrest of A431 human epidermoid cells and cells derived from multi-drug resistant tumors (10, EC(50)=7.8 nM). Competition for the colchicine binding site and pharmacokinetic properties of selected potent compounds were also investigated and are reported herein, along with structure-activity relationships for this novel series of antimitotic agents.     

SSB functions as a sliding platform that migrates on DNA via reptation

SSB proteins bind to and control the accessibility of single-stranded DNA (ssDNA), likely facilitated by their ability to diffuse on ssDNA. Using a hybrid single-molecule method combining fluorescence and force, we probed how proteins with large binding site sizes can migrate rapidly on DNA and how protein-protein interactions and tension may modulate the motion. We observed force-induced progressive unraveling of ssDNA from the SSB surface between 1 and 6 pN, followed by SSB dissociation at ～10 pN, and obtained experimental evidence of a reptation mechanism for protein movement along DNA wherein a protein slides via DNA bulge formation and propagation. SSB diffusion persists even when bound with RecO and at forces under which the fully wrapped state is perturbed, suggesting that even in crowded cellular conditions SSB can act as a sliding platform to recruit and carry its interacting proteins for use in DNA replication, recombination and repair.     

The crystal structure of BlmI as a model for nonribosomal peptide synthetase peptidyl carrier proteins

Carrier proteins (CPs) play a critical role in the biosynthesis of various natural products, especially in nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymology, where the CPs are referred to as peptidyl‐carrier proteins (PCPs) or acyl‐carrier proteins (ACPs), respectively. CPs can either be a domain in large multifunctional polypeptides or standalone proteins, termed Type I and Type II, respectively. There have been many biochemical studies of the Type I PKS and NRPS CPs, and of Type II ACPs. However, recently a number of Type II PCPs have been found and biochemically characterized. In order to understand the possible interaction surfaces for combinatorial biosynthetic efforts we crystallized the first characterized and representative Type II PCP member, BlmI, from the bleomycin biosynthetic pathway from Streptomyces verticillus ATCC 15003. The structure is similar to CPs in general but most closely resembles PCPs. Comparisons with previously determined PCP structures in complex with catalytic domains reveals a common interaction surface. This surface is highly variable in charge and shape, which likely confers specificity for interactions. Previous nuclear magnetic resonance (NMR) analysis of a prototypical Type I PCP excised from the multimodular context revealed three conformational states. Comparison of the states with the structure of BlmI and other PCPs reveals that only one of the NMR states is found in other studies, suggesting the other two states may not be relevant. The state represented by the BlmI crystal structure can therefore serve as a model for both Type I and Type II PCPs. Proteins 2014; 82:1210–1218. © 2013 Wiley Periodicals, Inc.     

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with k_cat ≈ 8 x 10⁻³ s⁻¹ and K_M < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower k_cat and a K_M ≈ 300 nM. The rate of ligation increased with addition of Mn²⁺, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.     

Ultrafast Redistribution of E. coli SSB along Long Single-Stranded DNA via Intersegment Transfer

Single-stranded DNA binding proteins (SSBs) selectively bind single-stranded DNA (ssDNA) and facilitate recruitment of additional proteins and enzymes to their sites of action on DNA. SSB can also locally diffuse on ssDNA, which allows it to quickly reposition itself while remaining bound to ssDNA. In this work, we used a hybrid instrument that combines single-molecule fluorescence and force spectroscopy to directly visualize the movement of Escherichia coli SSB on long polymeric ssDNA. Long ssDNA was synthesized without secondary structure that can hinder quantitative analysis of SSB movement. The apparent diffusion coefficient of E. coli SSB thus determined ranged from 70,000 to 170,000 nt²/s, which is at least 600 times higher than that determined from SSB diffusion on short ssDNA oligomers, and is within the range of values reported for protein diffusion on double-stranded DNA. Our work suggests that SSB can also migrate via a long-range intersegment transfer on long ssDNA. The force dependence of SSB movement on ssDNA further supports this interpretation.     

Molecular phylogeny of the tribe Candalidini (Lepidoptera: Lycaenidae): systematics,diversification and evolutionary history

The butterfly tribe Candalidini is geographically restricted to Australia and mainland New Guinea and its adjacent islands. With 60 species and subspecies, it represents a large radiation of Papilionoidea in the Australian region. Although the species-level taxonomy is relatively well understood, the number of genera is uncertain, varying from two to eight. We reconstructed the phylogeny of the Candalidini based on a 13-locus hybrid enrichment probe set (12.8 Kbp: COI, Thiolase, CAD, CAT, DDC, EF1-a, GAPDH, HCL, IDH, MDH, RPS2, RPS5, Wingless), including all previously recognized genera and 76% (28/37) of the species-level diversity of the tribe. Maximum likelihood analysis recovered the Candalidini as a strongly supported monophyletic group. In conjunction with morphological characters, the phylogeny provided a robust framework for a revised classification in which we recognize four genera, 37 species and 23 subspecies. The genus Nesolycaena Waterhouse & R.E. Turner is considered in synonymy with Candalides Hübner, and four other genera are not recognized, namely, Holochila C. Felder, Adaluma Tindale, Zetona Waterhouse and Microscena Tite. Of the four valid genera, the absimilis group (23 species) is placed in the newly described genus Eirmocides Braby, Espeland & Müller gen. nov. (type species Candalides consimilis Waterhouse). The erinus group (six species) is assigned to Erina Swainson, which is reinstated. Chrysophanus cyprotus Olliff is assigned to Cyprotides Tite, which is also reinstated as a monotypic genus. The remaining seven species are placed in Candalides sensu stricto. Overall, we propose 47 new nomenclatural changes at the species and subspecies levels, including the synonymy of Holochila biaka Tite as Eirmocides tringa biaka (Tite) syn. nov. et comb. nov. and recognition of Candalides hyacinthinus gilesi M.R. Williams & Bollam as a distinct species Erina gilesi (M.R. Williams & Bollam stat. rev. et comb. nov. A dated phylogeny using Bayesian inference in BEAST2 and biogeographical and habitat analyses based on the DEC model in BioGeoBEARS indicated that the ancestor of the Candalidini most likely evolved in rainforest habitats of the mesic biome in situ on the Australian plate of Southern Gondwana during the Eocene (c. 43 Ma). A major period of diversification occurred in the Miocene, which coincided with aridification of the Australian continent, followed by a further episode of radiation in montane New Guinea during the Plio-Pleistocene. This published work has been registered on ZooBank by the authors: Michael Braby: http://zoobank.org/urn:lsid:zoobank.org:author:4D3A7605-EBD0-40F6-A5F2-7F67F59E3D60 ; Marianne Espeland: http://zoobank.org/urn:lsid:zoobank.org:author:00D6F9F9-3902-4A8B-846F-720AB32922A6 ; Chris Müller: http://zoobank.org/urn:lsid:zoobank.org:author:15FE5F26-7596-46C2-9697-1FD92A692D0D ; http://zoobank.org/urn:lsid:zoobank.org:pub:47D5CA34-C294-4FBD-84B6-1C2A82B7CADF .     

Influence of treatment temperature on the genotoxic effects of cisplatin in CHO cells: cytotoxicity, mutagenicity and induction of lesions in DNA

In cells exposed in vitro to the cytotoxic and mutagenic antitumor drug cisplatin (cis-Pt(NH3)2Cl2), various adducts with nuclear DNA are formed. A comparative study was made of the influence of temperature variation during treatment of cultured Chinese hamster ovary (CHO) cells with cisplatin on cytotoxicity, mutation induction and Pt-DNA adduct formation. Before and after treatment (1 h at 32, 37 or 40 degrees C) cells were kept at 37 degrees C. Cytotoxicity increased with temperature; D0 values were 29.6 +/- 1.6, 21.1 +/- 1.2 and 11.4 +/- 0.6 microM at 32, 37 and 40 degrees C, respectively. Pt-DNA binding to DNA at 40 degrees C was 2.0 (+/- 0.3) times as high as at 32 degrees C. This factor remained practically constant over a 24-h post-treatment incubation of the cells, during which about 60% of DNA-bound Pt were removed. As the increase in cytotoxicity between 32 and 40 degrees C was roughly in proportion to that in Pt binding, no substantial changes in the spectrum of adducts appeared to occur. The induction of DNA interstrand cross-links, studied at 32 and 40 degrees C, varied linearly with dose. Influence of temperature on cross-link formation was comparable to that on total Pt binding. Amounts of cross-links highly increased during 24 h after treatment. Plots of cross-links against survival after treatments at 32 and 40 degrees C almost coincided. Induction of 6-thioguanine-resistant (HGPRT) mutants at various cisplatin concentrations did not show a clear temperature dependency. Consequently, equitoxic treatments were significantly more mutagenic at 32 degrees C than at 40 degrees C, the opposite of what has been reported for E. coli.