Transformations of Group 7 Carbonyl Complexes: Possible Intermediates in a Homogeneous Syngas Conversion Scheme

A variety of C−H and C−C bond forming reactions of group 7 carbonyl complexes have been studied as potential steps in a homogeneously catalyzed conversion of syngas to C_(2+) compounds. The metal formyl complexes M(CO)_3(PPh_3)_2(CHO) (M = Mn, Re) are substantially stabilized by coordination of boranes BX_3 (X = F, C_6F_5) in the form of novel boroxycarbene complexes M(CO)_3(PPh_3)_2(CHOBX_3), but these boron-stabilized carbenes do not react with hydride sources to undergo further reduction to metal alkyls. The related manganese methoxycarbene cations [Mn(CO)_(5−x)(PPh_3)_x(CHOMe)]+ (x = 1 or 2), obtained by methylation of the formyls, do react with hydrides to form methoxymethyl complexes, which undergo further migratory insertion under an atmosphere of CO. The resulting acyls, cis- and trans-Mn(PPh_3)(CO)_4(C(O)CH_2OMe), can be alkylated to form the cationic carbene complex [Mn(PPh_3)(CO)_4(C(OR)CH_2OMe)]^+, which undergoes a 1,2 hydride shift to form 1,2-dialkoxyethylene, which is displaced from the metal, releasing triflate or diethyl ether adducts of [Mn(PPh_3)(CO)_4]^+. The acyl can also be protonated with HOTf to form a hydroxycarbene complex, which rearranges to Mn(PPh_3)(CO)_4(CH_2COOMe) and is protonolyzed to yield methyl acetate and [Mn(PPh_3)(CO)_4]^+; addition of L (L = PPh_3, CO) to the manganese cation regenerates [Mn(PPh_3)(CO)_4(L)]^+. Since the original formyl complex can be obtained by the reaction of [Mn(PPh_3)(CO)_5]^+ with [PtH(dmpe)_2]^+, which in turn can be generated from H_2, this set of transformations amounts to a stoichiometric cycle for selectively converting H_2 and CO into a C_2 compound under mild conditions

Development of a Tunable Mammalian Protein Expression System and an Investigation of Promoter Interference in Three Promoters Often Utilized in the Production of Biopharmaceuticals

Cell line engineering strategies for improved biopharmaceutical production in mammalian cells often involve the expression of one/multiple genes to try and improve the cellular processes involved in recombinant protein production. Most strategies are relatively simple, involving the use of a strong constitutive promoter for expression of one or more proteins to help increase production. Results often vary and can be cell line and product specific and mean a generic strategy is unlikely to be found. There is a need for more sophisticated expression systems which can express multiple genes but in a controlled fashion and tuned to meet the needs of a specific product. This thesis can be split into two distinct parts but both concern the expression of multiple genes in mammalian cells and recombinant protein production. A tunable mammalian expression system for multi-gene engineering composed of elements of the mammalian unfolded protein response has been developed. ATF6 (activating transcription factor 6) and its binding element (ERSE – ER stress response element) were used to control the expression of the reporter proteins SEAP (secreted alkaline phosphatase) and GFP (green fluorescent protein). By expressing different amounts of ATF6 and by inserting different numbers of ERSEs upstream of a SV40 (Simian virus 40) promoter, driving SEAP/GFP gene transcription, the level of reporter protein expression could be manipulated in a controlled fashion. The system was capable of controlled/tunable expression of both reporter proteins when expressed alone and when they were co-expressed. This a novel use for ATF6 and ERSE and the first step towards the development of a tunable mammalian expression system for multi-gene engineering. This system could also be easily modified to include or use different transcription factors and binding sites as well as having the potential to use completely synthetic components. This work also showed that the presence of ‘promoter interference’ (the negative influence of one promoter on another) could be used to our advantage to increase the range of expression. The SV40 early, human CMV (cytomegalovirus) major immediate-early and human EF1α (elongation factor 1 alpha) are constitutive promoters frequently used in recombinant protein production. The former being used mainly for expression of selection genes and the latter two for strong expression of recombinant proteins. The differences in the strengths of the promoters was demonstrated in CHO (Chinese hamster ovary) cells (CMV > EF1α > SV40) and also their abilities to negatively affect the expression from a co-expressed promoter. The negative influence of one promoter on another is termed ‘promoter interference’. The CMV promoter was shown to have the greatest negative effect on expression from another promoter, decreasing both SEAP mRNA and protein expression, while the SV40 had the least. SEAP expression from the SV40 was reduced the most by the presence of a competing promoter. The level of interference inflicted by a competing promoter (CMV > EF1α > SV40) seemed to be relative to its strength. This is the first time these three important promoters have been compared in a way which not only demonstrates their relative strengths but also their ability to interfere with another promoter when present in the same transient expression system. This also has implications for their use in multi-gene engineering strategies if there is a need for controlled/tunable expression of multiple genes. The work with ATF6 and ERSE showed how ‘promoter interference’ could be used to our advantage and not necessarily be just a negative occurrence. One hypothesis for why promoter interference occurs is there is competition between promoters for shared transcription factors (TFs). The promoters were analysed for potential transcription factor binding sites (TFBSs) using the programs MatInspector and ModelInspector. The analysis showed that the SV40 promoter had the least number and variety of potential TFBSs. Both the CMV and EF1α had greater numbers and variety of potential TFBSs. All three promoters had common potential TFBSs but the SV40 promoter shared a greater proportion of its sites with the other two promoters. The number of potential TFBSs and the proportion which were shared reflected both the strength and the ability of a promoter to interfere with another. All three contained TFBSs for the SP1 (specificity protein 1) family of TFs and over-expression of SP1 counter-acted the effects of promoter interference showing that it can affect the expression of all three promoters. However, promoter interference will involve more than just a single TF and also more than just competition for transcriptional activators. This is the first time these three promoters have been compared in terms of the potential TFBSs they contain. The TFBS analysis highlighted the complexity in the control of these promoters and with the effects of promoter interference means that they will be ill suited for the controlled expression of multiple genes without modification. The work in this thesis was directed towards the controlled expression of multiple genes in mammalian cells for recombinant protein production. We have presented one novel way of controlling the expression of one/two genes with the rest of the thesis looking at the phenomenon of ‘promoter interference’ between three commonly used promoters. This thesis tries to highlight the importance of multi-gene expression systems as well showing that these three promoters may not be suitable without further modification and also the importance of considering promoter interactions when more than one is present in the same system. The switch to completely synthetic multi-gene control systems is something we envisage happening in the future as the complexities and capabilities of these systems grow

Genomic distribution of H3K9me2 and DNA methylation in a maize genome

DNA methylation and dimethylation of lysine 9 of histone H3 (H3K9me2) are two chromatin modifications that can be associated with gene expression or recombination rate. The maize genome provides a complex landscape of interspersed genes and transposons. The genome-wide distribution of DNA methylation and H3K9me2 were investigated in seedling tissue for the maize inbred B73 and compared to patterns of these modifications observed in Arabidopsis thaliana. Most maize transposons are highly enriched for DNA methylation in CG and CHG contexts and for H3K9me2. In contrast to findings in Arabidopsis, maize CHH levels in transposons are generally low but some sub-families of transposons are enriched for CHH methylation and these families exhibit low levels of H3K9me2. The profile of modifications over genes reveals that DNA methylation and H3K9me2 is quite low near the beginning and end of genes. Although elevated CG and CHG methylation are found within gene bodies, CHH and H3K9me2 remain low. Maize has much higher levels of CHG methylation within gene bodies than observed in Arabidopsis and this is partially attributable to the presence of transposons within introns for some maize genes. These transposons are associated with high levels of CHG methylation and H3K9me2 but do not appear to prevent transcriptional elongation. Although the general trend is for a strong depletion of H3K9me2 and CHG near the transcription start site there are some putative genes that have high levels of these chromatin modifications. This study provides a clear view of the relationship between DNA methylation and H3K9me2 in the maize genome and how the distribution of these modifications is shaped by the interplay of genes and transposons.The research was supported by a grant from the National Science Foundation (IOS-1237931) to MWV and NMS. This work also used resources or cyberinfrastructure provided by iPlant Collaborative. The iPlant Collaborative is funded by a grant from the National Science Foundation (DBI-0735191; www. iplantcollaborative.org). Start-up funds from the University of Georgia and a research grant from the National Science Foundation (IOS-1339194) to RJS supported aspects of this study

The UV, Optical, and IR Properties of SDSS Sources Detected by GALEX

We discuss the UV, optical, and IR properties of the SDSS sources detected by GALEX as part of its All-sky Imaging Survey Early Release Observations. Virtually all of the GALEX sources in the overlap region are detected by SDSS. GALEX sources represent ~2.5% of all SDSS sources within these fields and about half are optically unresolved. Most unresolved GALEX/SDSS sources are bright blue turn-off thick disk stars and are typically detected only in the GALEX near-UV band. The remaining unresolved sources include low-redshift quasars, white dwarfs, and white dwarf/M dwarf pairs, and these dominate the optically unresolved sources detected in both GALEX bands. Almost all the resolved SDSS sources detected by GALEX are fainter than the SDSS 'main' spectroscopic limit. These sources have colors consistent with those of blue (spiral) galaxies (u-r<2.2), and most are detected in both GALEX bands. Measurements of their UV colors allow much more accurate and robust estimates of star-formation history than are possible using only SDSS data. Indeed, galaxies with the most recent (<20 Myr) star formation can be robustly selected from the GALEX data by requiring that they be brighter in the far-UV than in the near-UV band. However, older starburst galaxies have UV colors similar to AGN, and thus cannot be selected unambiguously on the basis of GALEX fluxes alone. With the aid of 2MASS data, we construct and discuss median 10 band UV-optical-IR spectral energy distributions for turn-off stars, hot white dwarfs, low-redshift quasars, and spiral and elliptical galaxies. We point out the high degree of correlation between the UV color and the contribution of the UV flux to the UV-optical-IR flux of galaxies detected by GALEX.Comment: 35 pages, 11 figures, 3 tables; to appear in the AJ. PS with better figures available from http://www.astro.washington.edu/agueros/pub

Effect of obesity-linked FTO rs9939609 variant on physical activity and dietary patterns in physically active men and women

Single nucleotide polymorphisms (SNPs) in the fat mass and obesity-associated (FTO) locus are associated with obesity, but lifestyle factors may modulate the obesity risk related to FTO. This study examined the physical activity and dietary patterns of 528 physically active white men and women (mean(SD): 34.9(9.5) years, 26.6(4.3) kg·m-2) carrying different risk variants of the FTO SNP rs9939609. Sex, age and anthropometric measurements (stature, body mass, waist circumference) were self-reported using an online questionnaire, and body mass index and waist-to-height ratio were calculated. Physical activity and eating behaviour were assessed using the International Physical Activity Questionnaire and Three-Factor Eating Questionnaire (TFEQ), respectively. Body mass, body mass index, waist circumference and waist-to-height ratio were not significantly different between individuals expressing different FTO rs9939609 risk variants (all P≥0.66). The cohort was physically active (4516 (3043) total MET min·week-1), although homozygous risk allele carriers (AA) displayed higher TFEQ cognitive restraint compared with non-risk allele carriers (TT) (ES=0.33, P=0.03). In conclusion, obesity-related parameters were not different in physically active individuals expressing different risk variants of FTO rs9939609, although homozygous risk allele carriers exhibited higher cognitive restraint

The antecedents of biliary cancer: a primary care case–control study in the United Kingdom

In a case–control study using a large UK primary care database, we found that non-steroidal anti-inflammatory drugs had no protective effect against biliary carcinomas (cholangiocarcinoma and gall bladder cancer). Increased risks were observed for cigarette smoking, diabetes, gallstone disease and obesity

Essential Amino Acid Ingestion Facilitates Leucine Retention and Attenuates Myofibrillar Protein Breakdown following Bodyweight Resistance Exercise in Young Adults in a Home-Based Setting

Home-based resistance exercise (RE) has become increasingly prevalent, but its effects on protein metabolism are understudied. We tested the effect of an essential amino acid formulation (EAA+: 9 g EAAs, 3 g leucine) and branched-chain amino acids (BCAAs: 6 g BCAAs, 3 g leucine), relative to a carbohydrate (CHO) placebo, on exogenous leucine retention and myofibrillar protein breakdown following dynamic bodyweight RE in a home-based setting. Twelve recreationally active adults (nine male, three female) participated in a double-blind, placebo-controlled, crossover study with four trial conditions: (i) RE and EAA+ (EX-EAA+); (ii) RE and BCAAs (EX-BCAA); (iii) RE and CHO placebo (EX-CHO); and (iv) rest and CHO placebo (REST-CHO). Total exogenous leucine oxidation and retention (estimates of whole-body anabolism) and urinary 3-methylhistidine:creatinine ratio (3MH:Cr; estimate of muscle catabolism) were assessed over 5 h post-supplement. Total exogenous leucine oxidation and retention in EX-EAA+ and EX-BCAA did not significantly differ (p = 0.116) but were greater than EX-CHO (p < 0.01). There was a main effect of condition on urinary 3MH:Cr (p = 0.034), with post hoc analysis revealing a trend (p = 0.096) for reduced urinary 3MH:Cr with EX-EAA+ (32%) compared to EX-CHO. By direct comparison, urinary 3MH:Cr was significantly lower (23%) in EX-EAA+ than EX-BCAA (p = 0.026). In summary, the ingestion of EAA+ or BCAA provided leucine that was ~60% retained for protein synthesis following home-based bodyweight RE, but EAA+ most effectively attenuated myofibrillar protein breakdown

Combined In Silico, In Vivo, and In Vitro Studies Shed Insights into the Acute Inflammatory Response in Middle-Aged Mice

We combined in silico, in vivo, and in vitro studies to gain insights into age-dependent changes in acute inflammation in response to bacterial endotoxin (LPS). Time-course cytokine, chemokine, and NO2-/NO3- data from "middle-aged" (6-8 months old) C57BL/6 mice were used to re-parameterize a mechanistic mathematical model of acute inflammation originally calibrated for "young" (2-3 months old) mice. These studies suggested that macrophages from middle-aged mice are more susceptible to cell death, as well as producing higher levels of pro-inflammatory cytokines, vs. macrophages from young mice. In support of the in silico-derived hypotheses, resident peritoneal cells from endotoxemic middle-aged mice exhibited reduced viability and produced elevated levels of TNF-α, IL-6, IL-10, and KC/CXCL1 as compared to cells from young mice. Our studies demonstrate the utility of a combined in silico, in vivo, and in vitro approach to the study of acute inflammation in shock states, and suggest hypotheses with regard to the changes in the cytokine milieu that accompany aging. © 2013 Namas et al

Direct Multipixel Imaging and Spectroscopy of an Exoplant with a Solar Gravity Lens Mission

We report here on the results of our initial study of a mission to the deep outer regions of our solar system, with the primary mission objective of conducting direct megapixel high-resolution imag- ing and spectroscopy of a potentially habitable exoplanet by exploiting the remarkable optical properties of the SGL. Our main goal was not to study how to get there (although this was also addressed), but rather, to investigate what it takes to operate spacecraft at such enormous distances with the needed precision. Specifically, we studied i) how a space mission to the focal region of the SGL may be used to obtain high-resolution direct imaging and spectroscopy of an exoplanet by detecting, tracking, and studying the Einstein ring around the Sun, and ii) how such information could be used to detect signs of life on another planet