Foodplant Processing Adaptations in Four \u3ci\u3eHyalophora\u3c/i\u3e Species (Lepidoptera: Saturniidae): Regional and Taxonomic Specialization

To determine whether local populations of four Hyalophora species (Lepidoptera: Satumiidae) had improved survival or were physiologically adapted for rapid and/or efficient growth on their local hosts, a series of larval feeding studies were conducted using gravimetric techniques on several host plant species. Significantly better survival and growth performances were observed for H. columbia (a tamarack specialist) on its host, Larix laricina. Similarly, H. gloveri had the best growth performance on Elaeagnus angustifolia (its favorite) as did certain sympatric populations of H. cecropia on black cherry, Prunus serotina. Hyalophora gloveri and H. columbia are largely sympatric with Betula papyrifera and perform better than their allopatric congeners H. cecropia in the east and H. euryalus in the west. While survival of the tamarack specialist was poor, all three of the other North American Hyalophora species survived and grew very well on choke cherry, Prunus virginiana, which is sympatric with all four Hyalophora species. The extent to which these are genetically based adaptations is not known, nor are the specific mechanisms of biochemical adaptation involved in these differential performances of larvae

Novel nucleic acid molecules

The inventors have engineered novel DNA constructs for the expression of the tRNApyl genes in eukaryotic cells, especially mammalian cells, under new and improved promoter systems. The tRNApyl gene sequence possesses two internal regions that resemble an eukaryotic A box and B box, with the B box-like region more closely resembling a functional B box. Although previous attempts at reconstituting a consensus A- Box and B-box were unsuccessful as reported in Hancock et al (2010) and Mukai et al. (US 8,168,407), the inventors have now surprisingly found that the tRNApyl sequence can be altered to enable a functioning intragenic promoter and obtain a tRNApyl able to mediate efficient amber suppression in combination with WT pylRS. Such new tRNApyl gene can be used to generate highly active and stable cell lines for the incorporation of nnAAs into cells. The inventors have also found that the new modified tRNApyl gene containing a functional intragenic promoter element can be further improved by placing them downstream of the 5’ regulatory elements of genes expressed under type 4 promoters, thereby reconstituting a functional type 4 promoter element containing both extragenic and intragenic elements. The inventors have also surprisingly found that the WT tRNApyl gene can be expressed under transcriptional control of a tRNAglu gene and/or a tRNAasp gene, when said tRNAglu gene and/or tRNAasp gene is placed upstream of the tRNApyl gene and altered to lack the transcription termination sequence in order to effectively form a bicistronic message. DNA constructs bearing tandem repeats of novel tRNApyl genes of the invention have shown to lead to increased amber suppression

Improved Algorithms for Simulating Crystalline Membranes

The physics of crystalline membranes, i.e. fixed-connectivity surfaces embedded in three dimensions and with an extrinsic curvature term, is very rich and of great theoretical interest. To understand their behavior, numerical simulations are commonly used. Unfortunately, traditional Monte Carlo algorithms suffer from very long auto-correlations and critical slowing down in the more interesting phases of the model. In this paper we study the performance of improved Monte Carlo algorithms for simulating crystalline membrane, such as hybrid overrelaxation and unigrid methods, and compare their performance to the more traditional Metropolis algorithm. We find that although the overrelaxation algorithm does not reduce the critical slowing down, it gives an overall gain of a factor 15 over the Metropolis algorithm. The unigrid algorithm does, on the other hand, reduce the critical slowing down exponent to z apprx. 1.7.Comment: 14 pages, 1 eps-figur

Processing of N-Terminal Unnatural Amino Acids in Recombinant Human Interferon-β in Escherichia coli

Incorporation of unnatural amino acids into recombinant proteins represents a powerful tool for protein engineering and protein therapeutic development. While the processing of the N-terminal methionine (Met) residues in proteins is well studied, the processing of unnatural amino acids used for replacing the N-terminal Met remains largely unknown. Here we report the effects of the penultimate residue (the residue after the initiator Met) on the processing of two unnatural amino acids, L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG), at the N terminus of recombinant human interferon-β in E. coli. We have identified specific amino acids at the penultimate position that can be used to efficiently retain or remove N-terminal AHA or HPG. Retention of N-terminal AHA or HPG can be achieved by choosing amino acids with large side chains (such as Gln, Glu, and His) at the penultimate position, while Ala can be selected for the removal of N-terminal AHA or HPG. Incomplete processing of N-terminal AHA and HPG (in terms of both deformylation and cleavage) was observed with Gly or Ser at the penultimate position

Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects

Human cytomegalovirus (HCMV) infections of immunocompetent hosts are characterized by a dynamic, life-long interaction in which host immune responses, particularly of T cells, restrain viral replication and prevent disease but do not eliminate the virus or preclude transmission. Because HCMV is among the largest and most complex of known viruses, the T cell resources committed to maintaining this balance have never been characterized completely. Here, using cytokine flow cytometry and 13,687 overlapping 15mer peptides comprising 213 HCMV open reading frames (ORFs), we found that 151 HCMV ORFs were immunogenic for CD4+ and/or CD8+ T cells, and that ORF immunogenicity was influenced only modestly by ORF expression kinetics and function. We further documented that total HCMV-specific T cell responses in seropositive subjects were enormous, comprising on average ∼10% of both the CD4+ and CD8+ memory compartments in blood, whereas cross-reactive recognition of HCMV proteins in seronegative individuals was limited to CD8+ T cells and was rare. These data provide the first glimpse of the total human T cell response to a complex infectious agent and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans