Gold nanorods targeted to delta opioid receptor: plasmon-resonant contrast and photothermal agents

Molecularly targeted gold nanorods were investigated for applications in both diagnostic imaging and disease treatment with cellular resolution. The nanorods were tested in two genetically engineered cell lines derived from the human colon carcinoma HCT-116, a model for studying ligand-receptor interactions. One of these lines was modified to express delta opioid receptor (deltaOR) and green fluorescent protein, whereas the other was receptor free and expressed a red fluorescent protein, to serve as the control. Deltorphin, a high-affinity ligand for deltaOR, was stably attached to the gold nanorods through a thiol-terminated linker. In a mixed population of cells, we demonstrated selective imaging and destruction of receptor-expressing cells while sparing those cells that did not express the receptor. The molecularly targeted nanorods can be used as an in vitro ligand-binding and cytotoxic treatment assay platform and could potentially be applied in vivo for diagnostic and therapeutic purposes with endoscopic technology.     

Development of a 16S rRNA gene-based prototype microarray for the detection of selected actinomycetes genera

Actinomycetes are known for their secondary metabolites, which have been successfully used as drugs in human and veterinary medicines. However, information on the distribution of this group of Gram-positive bacteria in diverse ecosystems and a comprehension of their activities in ecosystem processes are still scarce. We have developed a 16S rRNA-based taxonomic microarray that targets key actinomycetes at the genus level. In total, 113 actinomycete 16S rRNA probes, corresponding to 55 of the 202 described genera, were designed. The microarray accuracy was evaluated by comparing signal intensities with probe/target-weighted mismatch values and the Gibbs energy of the probe/target duplex formation by hybridizing 17 non-actinomycete and 29 actinomycete strains/clones with the probe set. The validation proved that the probe set was specific, with only 1.3% of false results. The incomplete coverage of actinomycetes by a genus-specific probe was caused by the limited number of 16S rRNA gene sequences in databases or insufficient 16S rRNA gene polymorphism. The microarray enabled discrimination between actinomycete communities from three forest soil samples collected at one site. Cloning and sequencing of 16S rRNA genes from one of the soil samples confirmed the microarray results. We propose that this newly constructed microarray will be a valuable tool for genus-level comparisons of actinomycete communities in various ecological conditions.     

Strong dominance of functional alleles over gene deletions in both intensely growing and deeply starved yeast cells

Previous studies with diploid yeast have shown that the deletion of one allele at a single locus typically has little impact on fitness under conditions promoting fast growth. Here, we confirm and quantify this finding. The strong dominance of functional over nonfunctional alleles is predicted by the metabolic control theory which assumes that the cell is a system of metabolic fluxes and that the total metabolic rate is equivalent to fitness. To test whether these requirements are critical, we tested dominance under conditions of long‐term starvation when metabolism is low and thus the metabolic activities of proteins are likely inadequate or imbalanced. More fundamentally, the central assumption of the model, that high metabolic rate translates into high fitness, appears implausible. Contrary to these conjectures, we found that the mean rate of survival of starving heterozygotes was affected only slightly more than was the mean rate of growth under good conditions. Under none of the two treatments the central prediction of the model, that fitness of heterozygous strains is higher for the enzymatic proteins than for nonenzymatic ones, was confirmed. Our data add to growing uncertainty whether the metabolic control theory is sufficient to explain the remarkable ubiquity of strong genetic dominance.     

Synthesis,receptor binding studies,optical spectroscopic and in silico structural characterization of morphiceptin analogs with cis‐4‐amino‐L‐proline residues

Three novel morphiceptin analogs, in which Pro in position 2 and/or 4 was replaced by cis‐4‐aminoproline connected with the preceding amino acid through the primary amino group, were synthesized. The opioid receptor affinities, functional assay results, enzymatic degradation studies and experimental and in silico structural analysis of such analogs are presented. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Evolutionary conservation and network structure characterize genes of phenotypic relevance for mitosis in human

The impact of gene silencing on cellular phenotypes is difficult to establish due to the complexity of interactions in the associated biological processes and pathways. A recent genome-wide RNA knock-down study both identified and phenotypically characterized a set of important genes for the cell cycle in HeLa cells. Here, we combine a molecular interaction network analysis, based on physical and functional protein interactions, in conjunction with evolutionary information, to elucidate the common biological and topological properties of these key genes. Our results show that these genes tend to be conserved with their corresponding protein interactions across several species and are key constituents of the evolutionary conserved molecular interaction network. Moreover, a group of bistable network motifs is found to be conserved within this network, which are likely to influence the network stability and therefore the robustness of cellular functioning. They form a cluster, which displays functional homogeneity and is significantly enriched in genes phenotypically relevant for mitosis. Additional results reveal a relationship between specific cellular processes and the phenotypic outcomes induced by gene silencing. This study introduces new ideas regarding the relationship between genotype and phenotype in the context of the cell cycle. We show that the analysis of molecular interaction networks can result in the identification of genes relevant to cellular processes, which is a promising avenue for future research.     

A comparative analysis of MC4R gene sequence, polymorphism, and chromosomal localization in Chinese raccoon dog and Arctic fox

Numerous mutations of the human melanocortin receptor type 4 (MC4R) gene are responsible for monogenic obesity, and some of them appear to be associated with predisposition or resistance to polygenic obesity. Thus, this gene is considered a functional candidate for fat tissue accumulation and body weight in domestic mammals. The aim of the study was comparative analysis of chromosome localization, nucleotide sequence, and polymorphism of the MC4R gene in two farmed species of the Canidae family, namely the Chinese raccoon dog (Nycterutes procyonoides procyonoides) and the arctic fox (Alopex lagopus). The whole coding sequence, including fragments of 3'UTR and 5'UTR, shows 89% similarity between the arctic fox (1276 bp) and Chinese raccoon dog (1213 bp). Altogether, 30 farmed Chinese raccoon dogs and 30 farmed arctic foxes were searched for polymorphisms. In the Chinese raccoon dog, only one silent substitution in the coding sequence was identified; whereas in the arctic fox, four InDels and two single-nucleotide polymorphisms (SNPs) in the 5'UTR and six silent SNPs in the exon were found. The studied gene was mapped by FISH to the Chinese raccoon dog chromosome 9 (NPP9q1.2) and arctic fox chromosome 24 (ALA24q1.2-1.3). The obtained results are discussed in terms of genome evolution of species belonging to the family Canidae and their potential use in animal breeding.     

Interspecific recombinant congenic strains between C57BL/6 and mice of the Mus spretus species: a powerful tool to dissect genetic control of complex traits

Complex traits are under the genetic control of multiple genes, often with weak effects and strong epistatic interactions. We developed two new collections of mouse strains to improve genetic dissection of complex traits. They are derived from several backcrosses of the Mus spretus SEG/Pas or STF/Pas strains on the C57BL/6J background. Each of the 55 interspecific recombinant congenic strains (IRCSs) carries up to eight SEG/Pas chromosomal segments with an average size of 11.7 Mb, totalizing 1.37% of the genome. The complete series covers 39.7% of the SEG/Pas genome. As a complementary resource, six partial or complete interspecific consomic strains were developed and increased genome coverage to 45.6%. To evaluate the usefulness of these strains for QTL mapping, 16 IRCSs were compared with C57BL/6J for seven hematological parameters. Strain 66H, which carries three SEG/Pas chromosomal segments, had lower red blood cell volume and higher platelet count than C57BL/6J. Each chromosomal segment was isolated in a congenic strain to evaluate individual effects. Congenic strains were combined to assess epistasis. Our data show that both traits were controlled by several genes with complex epistatic interactions. IRCSs are therefore useful to unravel QTL with small effects and gene-by-gene interactions.     

Whole cell cryo-electron tomography reveals distinct disassembly intermediates of vaccinia virus

At each round of infection, viruses fall apart to release their genome for replication, and then reassemble into stable particles within the same host cell. For most viruses, the structural details that underlie these disassembly and assembly reactions are poorly understood. Cryo-electron tomography (cryo-ET), a unique method to investigate large and asymmetric structures at the near molecular resolution, was previously used to study the complex structure of vaccinia virus (VV). Here we study the disassembly of VV by cryo-ET on intact, rapidly frozen, mammalian cells, infected for up to 60 minutes. Binding to the cell surface induced distinct structural rearrangements of the core, such as a shape change, the rearrangement of its surface spikes and de-condensation of the viral DNA. We propose that the cell surface induced changes, in particular the decondensation of the viral genome, are a prerequisite for the subsequent release of the vaccinia DNA into the cytoplasm, which is followed by its cytoplasmic replication. Generally, this is the first study that employs whole cell cryo-ET to address structural details of pathogen-host cell interaction.     

Production, purification and partial enzymatic and molecular characterization of a laccase from the wood-rotting ascomycete Xylaria polymorpha

The hard wood-colonizing ascomycete Xylaria polymorpha, that is seemingly lacking peroxidases, produces laccase as sole ligninolytic oxidoreductase. The fungus secreted the enzyme preferably during the growth in complex media based on tomato juice. Addition of 2,5-xylidine considerably stimulated laccase production (up to 14,000 U l⁻¹). The enzyme was purified to homogeneity by anion exchange and size exclusion chromatography and characterized by biochemical and molecular methods. Xylaria laccase has a molecular mass of 67 kDa, a pI of 3.1 and an absorption maximum at 605 nm that is characteristic for blue copper proteins. It oxidized all typical laccase substrates including ABTS, 2,6-dimethoxyphenol, guaiacol as well as syringaldazine (catalytic efficiencies 3 × 10³ to 7 × 10⁴ M⁻¹ s⁻¹). The deduced amino acid sequence of one amplified laccase gene sequence between the copper binding regions 1 and 3 showed a high level of identity to some other laccases from ascomycetes. Furthermore, the sequence of an internal peptide fragment of the purified laccase was identical with an amino acid sequence deduced from the nucleotide sequence of the laccase gene. Xylaria laccase was found to oxidize a non-phenolic β-O-4 lignin model compound in presence of 1-hydroxybenzotriazole into the corresponding keto-form. The results of this study show that – in addition to ligninolytic basidiomycetes – also wood-dwelling ascomycetes can produce high titers of laccase that may be involved in the oxidation of lignin.     

An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies

Background

Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method.H5 and H7 specific monoclonal antibodies were experimentally raised and used in the development of inhibition ELISAs for detection of serological response specifically directed against AIV subtypes H5 and H7. The ELISAs were evaluated with polyclonal chicken anti-AIV antibodies against AIV subtypes: H1N2, H5N2, H5N7, H7N1, H7N7, H9N9, H10N4 and H16N3.

Results

Both the H5 and H7 ELISA proved to have a high sensitivity and specificity and the ELISAs detected H5 and H7 antibodies earlier during experimental infection than the HI test did. The reproducibility of the ELISA’s performed at different times was high with Pearson correlation coefficients of 0.96-0.98.

Conclusions

The ELISAs are a potential alternative to the HI test for screening of large amounts of avian sera, although only experimental sera were tested in this study.