Conformational HER-2/neu B-cell epitope peptide vaccine designed to incorporate two native disulfide bonds enhances tumor cell binding and antitumor activities

Cancer vaccines designed to elicit an antibody response that target antigenic sites on a tumor antigen must closely mimic the three-dimensional structure of the corresponding region on the antigen. We have designed a complex immunogen derived from the extracellular domain of human HER-2/neu-(626-649) that represents a three-dimensional epitope. We have successfully introduced two disulfide bonds into this sequence, thereby recapitulating the natural disulfide pairings observed in the native protein. To evaluate the immunogenicity of the doubly cyclized disulfide-linked peptide versus the free uncyclized peptide we examined the induction of antibody responses in both inbred and outbred mice strains, with both constructs eliciting high titered antibodies. The disulfide-paired specific antibodies exhibited enhanced cross-reactivity to HER-2/neu expressed on BT-474 cell line as determined by flow cytometry. The antitumor activities of the disulfidepaired specific antibodies did not improve the in vitro growth inhibition of human breast cancer cells overexpressing HER-2, but showed superior antitumor responses in the context of ADCC and interferon-gamma induction. Inbred mice (FVB/n) vaccinated with the disulfide-paired epitope exhibited a statistically significant reduction in the development of exogenously administered tumors in vivo compared with mice receiving either the free uncyclized or the promiscuous T-cell epitope (MVF) control peptide (p = 0.001). This study demonstrates the feasibility and importance of designing conformational epitopes that mimic the tertiary structure of the native protein for eliciting biologically relevant anti-tumor antibodies. Such approaches are a prerequisite to the design of effective peptide vaccines.     

Synthesis of 9-[1-(substituted)-3-(phosphonomethoxy)propyl]adenine derivatives as possible antiviral agents

Acyclic N9 adenine nucleosides substituted at C-1' position were prepared by the Mitsunobu reaction of 1-tert-butyldimethylsilyl-4-pivaloylbutan-1,2,4-triol (5) with adenine. Pivaloyl hydroxyl was modified to the phosphonomethoxy derivatives, and the tert-butyldimethylsilyl hydroxyl was converted to methoxy, azido, amino, fluoro, and c-hydroxyethyl and was eliminated to give vinyl. The resulting phosphonic acids were converted to prodrugs also.     

Comparative Whole-Genome Analysis of Clinical Isolates Reveals Characteristic Architecture of Mycobacterium tuberculosis Pangenome

The tubercle complex consists of closely related mycobacterium species which appear to be variants of a single species. Comparative genome analysis of different strains could provide useful clues and insights into the genetic diversity of the species. We integrated genome assemblies of 96 strains from Mycobacterium tuberculosis complex (MTBC), which included 8 Indian clinical isolates sequenced and assembled in this study, to understand its pangenome architecture. We predicted genes for all the 96 strains and clustered their respective CDSs into homologous gene clusters (HGCs) to reveal a hard-core, soft-core and accessory genome component of MTBC. The hard-core (HGCs shared amongst 100% of the strains) was comprised of 2,066 gene clusters whereas the soft-core (HGCs shared amongst at least 95% of the strains) comprised of 3,374 gene clusters. The change in the core and accessory genome components when observed as a function of their size revealed that MTBC has an open pangenome. We identified 74 HGCs that were absent from reference strains H37Rv and H37Ra but were present in most of clinical isolates. We report PCR validation on 9 candidate genes depicting 7 genes completely absent from H37Rv and H37Ra whereas 2 genes shared partial homology with them accounting to probable insertion and deletion events. The pangenome approach is a promising tool for studying strain specific genetic differences occurring within species. We also suggest that since selecting appropriate target genes for typing purposes requires the expected target gene be present in all isolates being typed, therefore estimating the core-component of the species becomes a subject of prime importance.     

Magnetite compensates for the lack of a pilin‐associated c‐type cytochrome in extracellular electron exchange

Nanoscale magnetite can facilitate microbial extracellular electron transfer that plays an important role in biogeochemical cycles, bioremediation and several bioenergy strategies, but the mechanisms for the stimulation of extracellular electron transfer are poorly understood. Further investigation revealed that magnetite attached to the electrically conductive pili of Geobacter species in a manner reminiscent of the association of the multi‐heme c‐type cytochrome OmcS with the pili of Geobacter sulfurreducens. Magnetite conferred extracellular electron capabilities on an OmcS‐deficient strain unable to participate in interspecies electron transfer or Fe(III) oxide reduction. In the presence of magnetite wild‐type cells repressed expression of the OmcS gene, suggesting that cells might need to produce less OmcS when magnetite was available. The finding that magnetite can compensate for the lack of the electron transfer functions of a multi‐heme c‐type cytochrome has implications not only for the function of modern microbes, but also for the early evolution of microbial electron transport mechanisms.     

Shark bycatch in the experimental tuna longline fishery in Lakshadweep Sea,India

Bycatch from the experimental longline operations in the Lakshadweep Sea were studied. The experiments were conducted on converted Pablo boats, originally used for pole and line fishing operations, to capture skipjack tuna in the Lakshadweep Islands. The overall bycatch rate was very high, with a mean hooking rate of 8.05/1000 hooks compared to the targeted tuna catch (1.75/1000 hooks). Bycatch contributed 82.4% of the catch in comparison to the tuna (17.6%) Thunnus albacares, in the longline operations. Silky shark (Carcharhinus falciformes) with 89.9% was the dominant shark species followed by C. amblyrhynchos, Galeocerdo cuvier, Alopias pelagicus, Negapriion acutedens and Sphyrna lewinii with 4.7, 2.7, 1.4, 0.7 and 0.7%, respectively. Sharks contributed to 74.1% of the catch, followed by 15.7% sailfishes and 10.2% miscellaneous fishes. Higher bycatch rates were evident during evening hours compared to mornings, but the results were not significant statistically. Studies on the effects of depth on the overall fishing performance and species selectivity failed to establish any significant relationship at a depth range of 35–100 m. Soaking time had a significant effect on bycatch rates. The hooking rate of sharks declined with an increase in soaking time. Considering the high shark bycatch in the fishery, an accurate monitoring of the longline fish catches in the Lakshadweep waters is an important step towards ensuring the sustainability of other populations, especially sharks.     

Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export,PfEMP1 Display,and Survival of Malaria Parasites

The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum–infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target.     

Synthesis of N6-Substituted 9-[3-(Phosphonomethoxy)Propyl]Adenine Derivatives As Possible Antiviral Agents

A number of N ⁶ -substituted 9-[3-(phosphonomethoxy)propyl]adenine derivatives having hydroxymethyl at C-1′-position were prepared from the appropriate 6-chloroadenine derivative. The syntheses of the corresponding prodrugs of these compounds are also reported. These compounds showed poor activity against HCV in replicon assay.     

Sequence-specific 1H, 13C and 15N assignments of the phosphoesterase (PE) domain of Pseudomonas aeruginosa DNA ligase D (LigD)

DNA ligase D (LigD), consisting of polymerase, ligase and phosphoesterase domains, is the essential catalyst of the bacterial non-homologous end-joining pathway of DNA double-strand break repair. The phosphoesterase (PE) module performs manganese-dependent 3′-phosphomonoesterase and 3′-ribonucleoside resection reactions that heal broken ends in preparation for sealing. LigD PE exemplifies a structurally and mechanistically unique class of DNA end-processing enzymes. Here, we present the resonance assignments of the PE domain of Pseudomonas aeruginosa LigD comprising the N-terminal 177 residues.     

Benchmarking and analysis of protein docking performance in Rosetta v3.2

RosettaDock has been increasingly used in protein docking and design strategies in order to predict the structure of protein-protein interfaces. Here we test capabilities of RosettaDock 3.2, part of the newly developed Rosetta v3.2 modeling suite, against Docking Benchmark 3.0, and compare it with RosettaDock v2.3, the latest version of the previous Rosetta software package. The benchmark contains a diverse set of 116 docking targets including 22 antibody-antigen complexes, 33 enzyme-inhibitor complexes, and 60 'other' complexes. These targets were further classified by expected docking difficulty into 84 rigid-body targets, 17 medium targets, and 14 difficult targets. We carried out local docking perturbations for each target, using the unbound structures when available, in both RosettaDock v2.3 and v3.2. Overall the performances of RosettaDock v2.3 and v3.2 were similar. RosettaDock v3.2 achieved 56 docking funnels, compared to 49 in v2.3. A breakdown of docking performance by protein complex type shows that RosettaDock v3.2 achieved docking funnels for 63% of antibody-antigen targets, 62% of enzyme-inhibitor targets, and 35% of 'other' targets. In terms of docking difficulty, RosettaDock v3.2 achieved funnels for 58% of rigid-body targets, 30% of medium targets, and 14% of difficult targets. For targets that failed, we carry out additional analyses to identify the cause of failure, which showed that binding-induced backbone conformation changes account for a majority of failures. We also present a bootstrap statistical analysis that quantifies the reliability of the stochastic docking results. Finally, we demonstrate the additional functionality available in RosettaDock v3.2 by incorporating small-molecules and non-protein co-factors in docking of a smaller target set. This study marks the most extensive benchmarking of the RosettaDock module to date and establishes a baseline for future research in protein interface modeling and structure prediction.