Insect expression systems : improving intracellular and heterologous gene expression

Expression systems (ES) in a wide variety of biological systems are used to provide foreign protein. Protein production in mammalian cells is a labour-intensive and expensive process and insect cells have been used as cheaper alternatives. Insect ES are based on two types of vector; viral- and plasmid-based. The baculovirus ES (BES) offers high-level transient production of intracellular proteins. Yields of secreted and membrane-targeted proteins have until recently been relatively low, however, the development of a novel vector, lacking chitinase, has overcome these limitations. Plasmid-based vectors can be used for transient and stable expression in insect cells but most utilise the relatively weak Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate early-1 (ie-1) promoter, producing low-levels of intracellular recombinant protein. More recent vectors have used stronger promoters, however, compared with mammalian stable ES these are limited in use and most notable is the lack of an efficient inducible ES in insect cells. The work in this thesis describes methods, with which intracellular levels of foreign protein may be increased, while also developing the grounding work for the development of a fully functional insect tetracycline regulatory system. To improve stable and regulated expression of heterologous genes in insect cells, work was undertaken to compare and characterise the transcriptional activity of a range of promoters successfully used in insect and mammalian systems. Of the promoters tested, Orgyia pseudotsugata MNPV (Op) ie-2 and Bombyx morl actin 3 (BmA3+E) were found to be transcriptionally stronger than AcMNPV ie-1. Drosophila melanogaster metallothionein and actin 5.1 did not produce any detectable activity, and the Cytomegalovirus (CMV) ie promoter, although active, was weaker than AcMNPV ;e-1. Attempts to develop a new inducible ES for use in stable cell lines was based on modifying a mammalian tetracyclineinducible ES. The original CMVie promoters were replaced by the Bm A3+E and Op ie-2 promoters that had been shown to be optimal in insect cells. The Bm A3+E promoter was successfully used to express the tetracycline transcriptional activator protein. Studies also demonstrated that the constitutive transcriptional activity of Op ie-2, used to drive expression of a reporter gene, was successfully suppressed in normal medium. However, in the presence of the inducer, doxycycline, transcriptional activity of Op ie-2 was not activated. Work to elucidate why Op ie-2 remain repressed in the presence of the inducer, indicated that other, uncharacterised vector sequences may have interfered with the activation process. To determine whether stable insect cell lines could be used as an effective alternative to the BES for producing large quantities of intracellular foreign proteins, protein production from stable cell lines, using the Bm A3+E promoter, were compared to the BES using a range of reporter proteins. It was concluded that with vectors currently available, stable cell lines would not normally provide an effective alternative to the BES. However, a stable insect cell line expressing Discosoma red was used to develop methods for scaling-up continuous cultivation of cells in an open fermenter system. This stable cell line was successfully maintained without contamination or total loss of cell viability for 4 weeks. Improving intracellular expression of foreign genes using the BES, initially focused on investigating whether a baculovirus vector (BV) lacking chitinase could produce larger yields than normal BV. It was concluded that recombinant BV lackingchitinase could be used to improve intracellular levels of foreign protein, but demonstrated the importance of optimiSing production conditions for each recombinant protein (e.g., vector and cell line). Intracellular levels of foreign protein were further improved from the BES using different culturing methodologies (fermentation and shaker flask) and concluded that fermenters produced optimal conditions for intracellular protein production, probably due to maintaining a constant level of dissolved oxygen concentration during virus infection

Sensory-specific Appetition: Postingestive Detection of Glucose Rapidly Promotes Continued Consumption of a Recently Encountered Flavor

It is generally thought that macronutrients stimulate intake when sensed in the mouth (e.g., sweet taste) but as food enters the GI tract its effects become inhibitory, triggering satiation processes leading to meal termination. Here we report experiments extending recent work (see [1]) showing that under some circumstances nutrients sensed in the gut produce a positive feedback effect, immediately promoting continued intake. In one experiment, rats with intragastric (IG) catheters were accustomed to consuming novel flavors in saccharin daily while receiving water infused IG (5 ml/15 min). The very first time glucose (16% w/w) was infused IG instead of water, intake accelerated within 6 mins of infusion onset and total intake increased 29% over baseline. Experiment 2 replicated this stimulatory effect with glucose infusion but not fructose nor maltodextrin. Experiment 3 showed the immediate intake stimulation is specific to the flavor accompanying the glucose infusion. Rats were accustomed to flavored saccharin being removed and replaced with the same or a different flavor. When glucose infusion accompanied the first bottle, intake from the second bottle was stimulated only when it contained the same flavor, not when the flavor switched. Thus we confirm not only that glucose sensed postingestively can have a rapid, positive feedback effect (\u27appetition\u27 as opposed to \u27satiation\u27) but that it is sensory-specific, promoting continued intake of a recently encountered flavor. This sensory specific motivation may represent an additional psychobiological influence on meal size, and further, has implications for the mechanisms of learned flavor-nutrient associations

Optical Sky Brightness at Cerro Tololo Inter-American Observatory from 1992 to 2006

We present optical UBVRI sky brightness measures from 1992 through 2006. The data are based on CCD imagery obtained with the CTIO 0.9-m, 1.3-m, and 1.5-m telescopes. The B- and V-band data are in reasonable agreement with measurements previously made at Mauna Kea, though on the basis of a small number of images per year there are discrepancies for the years 1992 through 1994. Our CCD-based data are not significantly different than values obtained at Cerro Paranal. We find that the yearly averages of V-band sky brightness are best correlated with the 10.7-cm solar flux taken 5 days prior to the sky brightness measures. This implies an average speed of 350 km/sec for the solar wind. While we can measure an enhancement of the night sky levels over La Serena 10 degrees above the horizon, at elevation angles above 45 degrees we find no evidence that the night sky brightness at Cerro Tololo is affected by artificial light of nearby towns and cities.Comment: 24 pages, 5 figures, to be published in the June, 2007, issue of the Publications of the Astron. Society of the Pacifi

Electrochemical Quantification of D-Glucose during the Production of Bioethanol from Thermo-Mechanically Pre-treated Wheat Straw

Mechanical pre-treatment (disc refining) of wheat straw, at both atmospheric and elevated pressure, is shown to be an efficient process to access fermentable monosaccharides, with the potential to integrate within the infrastructure of existing first-generation bioethanol plants. The mild, enzymatic degradation of this sustainable lignocellulosic biomass affords ca. 0.10-0.13 g/g (dry weight) of D-glucose quantifiable voltammetrically in real time, over a two hundred-fold range in experimental laboratory scales (25 mL to 5.0 L), with pressure disc refining of the wheat straw enabling almost twice the amount of D-glucose to be generated during the hydrolysis stage than experiments using atmospheric refining (0.06 – 0.09 g/g dry weight). Fermentation of the resulting hydrolysate affords 0.08 – 0.10 g/g (dry weight) of ethanol over similar scales, with ethanol productivity at ca. 37 mg/(L h). These results demonstrate that minimal cellulose decomposition occurs during pressure refining of wheat straw, in contrast to hemicellulose, and suggest that the development of green, mechanochemical processes for the scalable and cost-effective manufacture of second-generation bioethanol requires improved cellulose decomposition

Limited antigenic diversity of Plasmodium falciparum apical membrane antigen 1 supports the development of effective multi-allele vaccines

BackgroundPolymorphism in antigens is a common mechanism for immune evasion used by many important pathogens, and presents major challenges in vaccine development. In malaria, many key immune targets and vaccine candidates show substantial polymorphism. However, knowledge on antigenic diversity of key antigens, the impact of polymorphism on potential vaccine escape, and how sequence polymorphism relates to antigenic differences is very limited, yet crucial for vaccine development. Plasmodium falciparum apical membrane antigen 1 (AMA1) is an important target of naturally-acquired antibodies in malaria immunity and a leading vaccine candidate. However, AMA1 has extensive allelic diversity with more than 60 polymorphic amino acid residues and more than 200 haplotypes in a single population. Therefore, AMA1 serves as an excellent model to assess antigenic diversity in malaria vaccine antigens and the feasibility of multi-allele vaccine approaches. While most previous research has focused on sequence diversity and antibody responses in laboratory animals, little has been done on the cross-reactivity of human antibodies.MethodsWe aimed to determine the extent of antigenic diversity of AMA1, defined by reactivity with human antibodies, and to aid the identification of specific alleles for potential inclusion in a multi-allele vaccine. We developed an approach using a multiple-antigen-competition enzyme-linked immunosorbent assay (ELISA) to examine cross-reactivity of naturally-acquired antibodies in Papua New Guinea and Kenya, and related this to differences in AMA1 sequence.ResultsWe found that adults had greater cross-reactivity of antibodies than children, although the patterns of cross-reactivity to alleles were the same. Patterns of antibody cross-reactivity were very similar between populations (Papua New Guinea and Kenya), and over time. Further, our results show that antigenic diversity of AMA1 alleles is surprisingly restricted, despite extensive sequence polymorphism. Our findings suggest that a combination of three different alleles, if selected appropriately, may be sufficient to cover the majority of antigenic diversity in polymorphic AMA1 antigens. Antigenic properties were not strongly related to existing haplotype groupings based on sequence analysis.ConclusionsAntigenic diversity of AMA1 is limited and a vaccine including a small number of alleles might be sufficient for coverage against naturally-circulating strains, supporting a multi-allele approach for developing polymorphic antigens as malaria vaccines

The 31 Deg2^2 Release of the Stripe 82 X-ray Survey: The Point Source Catalog

We release the next installment of the Stripe 82 X-ray survey point-source catalog, which currently covers 31.3 deg$^2$ of the Sloan Digital Sky Survey (SDSS) Stripe 82 Legacy field. In total, 6181 unique X-ray sources are significantly detected with {\it XMM-Newton} ($>5\sigma$) and {\it Chandra} ($>4.5\sigma$). This catalog release includes data from {\it XMM-Newton} cycle AO 13, which approximately doubled the Stripe 82X survey area. The flux limits of the Stripe 82X survey are $8.7\times10^{-16}$ erg s$^{-1}$ cm$^{-2}$, $4.7\times10^{-15}$ erg s$^{-1}$ cm$^{-2}$, and $2.1\times10^{-15}$ erg s$^{-1}$ cm$^{-2}$ in the soft (0.5-2 keV), hard (2-10 keV), and full bands (0.5-10 keV), respectively, with approximate half-area survey flux limits of $5.4\times10^{-15}$ erg s$^{-1}$ cm$^{-2}$, $2.9\times10^{-14}$ erg s$^{-1}$ cm$^{-2}$, and $1.7\times10^{-14}$ erg s$^{-1}$ cm$^{-2}$. We matched the X-ray source lists to available multi-wavelength catalogs, including updated matches to the previous release of the Stripe 82X survey; 88\% of the sample is matched to a multi-wavelength counterpart. Due to the wide area of Stripe 82X and rich ancillary multi-wavelength data, including coadded SDSS photometry, mid-infrared {\it WISE} coverage, near-infrared coverage from UKIDSS and VHS, ultraviolet coverage from {\it GALEX}, radio coverage from FIRST, and far-infrared coverage from {\it Herschel}, as well as existing $\sim$30\% optical spectroscopic completeness, we are beginning to uncover rare objects, such as obscured high-luminosity AGN at high-redshift. The Stripe 82X point source catalog is a valuable dataset for constraining how this population grows and evolves, as well as for studying how they interact with the galaxies in which they live.Comment: accepted for publication in ApJ; 23 pages (emulateapj

A photonic platform for donor spin qubits in silicon

Donor spins in silicon are highly competitive qubits for upcoming quantum technologies, offering complementary metal-oxide semiconductor compatibility, coherence (T2) times of minutes to hours, and simultaneous initialization, manipulation, and readout fidelities near ~99.9%. This allows for many quantum error correction protocols, which will be essential for scale-up. However, a proven method of reliably coupling spatially separated donor qubits has yet to be identified. We present a scalable silicon-based platform using the unique optical properties of “deep” chalcogen donors. For the prototypical 77Se+ donor, we measure lower bounds on the transition dipole moment and excited-state lifetime, enabling access to the strong coupling limit of cavity quantum electrodynamics using known silicon photonic resonator technology and integrated silicon photonics. We also report relatively strong photon emission from this same transition. These results unlock clear pathways for silicon-based quantum computing, spin-to-photon conversion, photonic memories, integrated single-photon sources, and all-optical switches