Simultaneous Surface Display and Secretion of Proteins from Mammalian Cells Facilitate Efficient in Vitro Selection and Maturation of Antibodies

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.     

Low-Resolution Structure of Vaccinia Virus DNA Replication Machinery

Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the Orthopoxvirus genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages.     

Endogenous hormone profiles during early seed development of C. arietinum and C. anatolicum

Cicer anatolicum, a perennial species, has ascochyta blight resistance superior to that found in the cultivated chickpea. However, hybridization barriers during early stages of embryo development curtail access to this trait. Since hormones play an essential role in early embryo development, we have determined the hormone profiles of 4-, 8-, and 12-day old seeds from a Canadian chickpea (Cicer arietinum L.) cv. CDC Xena, from Indian cvs. Swetha and Bharati, and from a perennial accession of C. anatolicum (PI 383626). Indole-3-acetic acid content peaked on day 4 in CDC Xena, on day 8 in both Indian cultivars but only on day 12 in C. anatolicum. The cytokinins, isopentenyladenosine (iPA) and trans zeatin riboside (tZR) were predominant in CDC Xena and Swetha seeds on day 4, whereas cis zeatin riboside was the major component in Bharati. In C. anatolicum, iPA maxed out on day 4 and tZR on day 12. The bioactive gibberellin GA₁ spiked on day 4 in CDC Xena and Bharati, on day 8 in Swetha but only on day 12 in C. anatolicum. Eight-day old seeds had the highest abscisic acid content in the cultivars but spiked on day 12 in the perennial species. The hormone profiles of the perennial species showed delayed spikes in all four hormone groups indicating that there is a mismatch in the hormone requirements of the different embryos. Improving synchronization of early seed hormone profiles of cultivated and perennial chickpea should improve interspecific hybrid production.     

DNA barcoding survey of Trichoderma diversity in soil and litter of the Colombian lowland Amazonian rainforest reveals Trichoderma strigosellum sp. nov. and other species

The diversity of Trichoderma (Hypocreales, Ascomycota) colonizing leaf litter as well as the rhizosphere of Garcinia macrophylla (Clusiaceae) was investigated in primary and secondary rain forests in Colombian Amazonia. DNA barcoding of 107 strains based on the internal transcribed spacers 1 and 2 (ITS1 and 2) of the ribosomal RNA gene cluster and the partial sequence of the translation elongation factor 1 alpha (tef1) gene revealed that the diversity of Trichoderma was dominated (71 %) by three common cosmopolitan species, namely Trichoderma harzianum sensu lato (41 %), Trichoderma spirale (17 %) and Trichoderma koningiopsis (13 %). Four ITS 1 and 2 phylotypes (13 strains) could not be identified with certainty. Multigene phylogenetic analysis and phenotype profiling of four strains with an ITS1 and 2 phylotype similar to Trichoderma strigosum revealed a new sister species of the latter that is described here as Trichoderma strigosellum sp. nov. Sequence similarity searches revealed that this species also occurs in soils of Malaysia and Cameroon, suggesting a pantropical distribution.     

Palynological evaluation of selected honeys from Romania

Fifty-four honey samples collected over two consecutive harvest seasons (2009–2010) from different floristic areas of Romania were analysed. A melissopalynological analysis including qualitative and quantitative analyses was carried out in order to identify the principal pollen types in Romanian honeys and therefore, the important plants exploited by Apis mellifera in this country. Seventy-seven pollen types from 35 botanical families were identified. The main pollen forms were: Brassica napus-type, Tilia, Helianthus annuus, Robinia pseudoacacia, Prunus, Castanea sativa, Fragaria-type and Plantago-type. Honey samples were classified as: acacia honey (Robinia pseudoacacia), lime honey (Tilia), rape honey (Brassica napus-type), sunflower honey (Helianthus annuus), cherry honey (Prunus). Three of them were honeydew honeys whereas the others were all polyfloral. The results from this study can be used as a palynological baseline data of Romanian artisanal honeys.     

Spore morphology of the north Asian members of Cystopteridaceae

Spores of Cystopteridaceae from northern Asia were examined using scanning electron microscopy. To evaluate the utility of spore morphology in the taxonomy of each genus, we examined spores of 14 species: seven species each of Gymnocarpium and Cystopteris. Among these are 12 species occurring in northern Asia and two species from other regions for comparative studies. The study focused particularly on perispore characters and spore size. Spores of all species examined are monolete, bean-shaped, with a range in spore size of 26–56 × 18–37 μm for Cystopteris and 25–48 × 16–34 μm for Gymnocarpium. The perispore is morphologically diverse within Cystopteris, but less so within Gymnocarpium. The perispore of the Cystopteris spores is characterised by folds and spines that are separate or form complex sculptural elements. Sacci, ridges and flanges, sometimes on the same spore, are characteristic of the perispore of Gymnocarpium. Spores have straight laesura over which the perispore forms a crest. The crest represents a high and flat fold, which is entire, foveolate or reticulate.     

Opioid receptor selectivity profile change via isosterism for 14-O-substituted naltrexone derivatives

Isosterism is commonly used in drug discovery and development to address stability, selectivity, toxicity, pharmacokinetics, and efficacy issues. A series of 14-O-substituted naltrexone derivatives were identified as potent mu opioid receptor (MOR) antagonists with improved selectivity over the kappa opioid receptor (KOR) and the delta opioid receptor (DOR), compared to naltrexone. Since esters are not metabolically very stable under typical physiological conditions, their corresponding amide analogs were thus synthesized and biologically evaluated. Unlike their isosteres, most of these novel ligands seem to be dually selective for the MOR and the KOR over the DOR. The restricted flexibility of the amide bond linkage might be responsible for their altered selectivity profile. However, the majority of the 14-N-substituted naltrexone derivatives produced marginal or no MOR stimulation in the ³⁵S-GTP[γS] assay, which resembled their ester analogs. The current study thus indicated that the 14-substituted naltrexone isosteres are not bioisosteres since they have distinctive pharmacological profile with the regard to their opioid receptor binding affinity and selectivity.     

Fine mechanisms of the interaction of silver nanoparticles with the cells of Salmonella typhimurium and Staphylococcus aureus

Silver nanoparticles possess antibacterial effect for various bacteria; however mechanisms of the interaction between Ag-NPs and bacterial cells remain unclear. The aim of our study was to obtain direct evidence of Ag-NPs penetration into cells of Gram-negative bacterium S. typhimurium and Gram-positive bacterium S. aureus, and to study cell responses to Ag-NPs. The Ag-NPs (most 8–10 nm) were obtained by gas-jet method. S. typhimurium (7.81 × 10⁷ CFU), or S. aureus (8.96 × 10⁷ CFU) were treated by Ag-NPs (0.05 mg/l of silver) in orbital shaker at 190 rpm, 37 °C. Bacteria were sampled at 0.5, 1, 1.5, 2, 5 and 23 h of the incubation for transmission electron microscopy of ultrathin sections. The Ag-NPs adsorbed on outer membrane of S. typhimurium and cell wall of S. auereus; penetrated and accumulated in cells without aggregation and damaging of neighboring cytoplasm. In cells of S. aureus Ag-NPs bound with DNA fibers. Cell responses to Ag-NPs differed morphologically in S. typhimurium and S. aureus, and mainly were presented by damage of cell structures. The cytoplasm of S. aureus became amorphous, while S. typhimurium showed lumping and lysis of cytoplasm which led to formation of “empty” cells. Other difference was fast change of cell shape in S. typhimurium, and late deformation of S. aureus cells. The obtained results showed how different could be responses induced by the same NPs in relatively simple prokaryotic cells. Evidently, Ag-NPs directly interact with macromolecular structures of living cells and are exert an active influence on their metabolism.     

Choosing the right antibody for resistin-like molecule (RELM/FIZZ) family members

The family of resistin-like molecules (RELM), also known as found in inflammatory zone (FIZZ), consists of four members in mouse (RELMα/FIZZ1/HIMF, RELMβ/FIZZ2, Resistin/FIZZ3, and RELMγ/FIZZ4) and two members in human (resistin and RELMβ). The importance of these proteins in many aspects of physiology and pathophysiology, especially inflammatory processes, is rapidly evolving in the literature, and many investigators are beginning to work in this field. Most published studies focus on only one isoform, do not evaluate other isoforms that might be present, and have not tested for the specificity of the antibody used. Because RELM isoforms have high sequence and structural similarity and both distinct and overlapping functions, it is important to use a specific antibody to distinguish each isoform in the study. We constructed and established HEK 293 cell lines that constitutively express each isoform. Using these cell lines, we determined the specificity of antibodies (both commercially available and laboratory-made) to each isoform by Western blot and immunofluorescence. Some of the antibodies showed specificity in Western blotting but were not applicable in immunofluorescence. Others showed cross reactivity in Western blot assays. Our results indicate that RELM antibody specificity should be taken into account when using them in research and interpreting data obtained with them.