A high-yielding serum-free, suspension cell culture process to manufacture recombinant adenoviral vectors for gene therapy

We have developed an efficient, reproducible, and scaleable cell culture process for a recombinant adenoviral vector expressing therapeutic transgenes for clinical trials. HEK 293 cells – which support the propagation of E1 deficient adenovirus – were first adapted to serum free media and suspension growth. Subsequent studies focused on the infection, virus production and harvest from suspension culture bioreactors. Future studies are planned to address the kinetics of adenovirus production in HEK 293 as well as in other cell lines. This revised version was published online in August 2006 with corrections to the Cover Date.     

Flexibility in the donor substrate specificity of {beta} 1,4-galactosyltransferase: application in the synthesis of complex carbohydrates

Biosynthetically, bovine N-acetylglucosainine ß 1,4-galacto-syltransferase(GalT) catalyses the transfer of galactosyl residues from UDP-Galto the 4-position of GlcNAc units, resulting in the productionof N-acetyllactosamine sequences. UDP-Glc and UDP-GalNAc werealso found to act as donors for this enzyme, allowing the preparationof ßGlc(14)-ßGlcNAc and ßGalNAc(14)ßGlcNActerminating structures on the milligram scale. GalT could thusbe used to add ßGalNAc to ßGlcNAc(12)Manterminating structures, converting them to the ßGalNAc(14)ßGlcNAc(12)Mansequences found on glycoprotein hormones. GalT did not transferGlcNAc residues from UDP-GlcNAc, but it could utilize UDP-GlcNH₂as a donor. Synthesis of ßGlcNAc(14)ßGlcNAcsequences could therefore be accomplished by transfer of GlcNH₂from its UDP derivative, followed by N-acetylation of the productamino-disaccharide using acetic anhydride in methanol. The productsof the enzymatic reactions were characterized by ¹H-NMR-spectroscopyand fast-atom bombardment mass spectrometry. This work expandsthe scope of the combined chemical-enzymatic synthesis of complexcarbohydrates, using glycosyltrans-ferases, to the productionof oligosaccharides different from those for which these enzymeswere designed. These unnatural reactions should find applicationin glycoprotein and glycolipid remodelling. galactosyltransferase chemica1-enzymatic synthesis of oligosaccharides oligosaccharide analogues sugar-nucleotide analogues carbohydrate remodelling     

Edible zombis: fresh fish and the industry of cosmetic corpses

Doing research on fishery commodities in Portugal led us to an enigma: for a dead fish to be fresco (fresh) it must be alive. This paradox manifests at a popular, commercial, and legal level. It denotes the interruption of the difference between being dead and being alive in the commodity form. In Portugal, we suggest, the commercialization of peixe fresco (fresh fish) is based on the production and consumption of edible ‘zombis’: seafood corpses technologically and symbolically crafted as undead. An open concept, ‘edible zombis’ is part of an experimental vocabulary that foregrounds the productive agency of undeadness, both biological and commercial, in the seafood economic complex. It relates to the ordinary practice of necromancy in the commodity-based world. Edible zombis are commodity fetishes that fetishize their producers and consumers, suspending them from the capitalist system in which they live.     

Cyclic lipopeptide-producing Pseudomonas koreensis group strains dominate the cocoyam rhizosphere of a Pythium root rot suppressive soil contrasting with P. putida prominence in conducive soils

Pseudomonas isolates from tropical environments have been underexplored and may form an untapped reservoir of interesting secondary metabolites. In this study, we compared Pseudomonas and cyclic lipopeptide (CLP) diversity in the rhizosphere of a cocoyam root rot disease (CRRD) suppressive soil in Boteva, Cameroon with those from four conducive soils in Cameroon and Nigeria. Compared with other soils, Boteva andosols were characterized by high silt, organic matter, nitrogen and calcium. Besides, the cocoyam rhizosphere at Boteva was characterized by strains belonging mainly to the P. koreensis and P. putida (sub)groups, with representations in the P. fluorescens, P. chlororaphis, P. jessenii and P. asplenii (sub)groups. In contrast, P. putida isolates were prominent in conducive soils. Regarding CLP diversity, Boteva was characterized by strains producing 11 different CLP types with cocoyamide A producers, belonging to the P. koreensis group, being the most abundant. However, putisolvin III-V producers were the most dominant in the rhizosphere of conducive soils in both Cameroon and Nigeria. Furthermore, we elucidated the chemical structure of putisolvin derivatives—putisolvin III-V, and described its biosynthetic gene cluster. We show that high Pseudomonas and metabolic diversity may be driven by microbial competition, which likely contributes to soil suppressiveness to CRRD.     

Transforming Growth Factor Beta Receptor 1 Is Increased following Abstinence from Cocaine Self-Administration,but Not Cocaine Sensitization

The addicted phenotype is characterized as a long-lasting, chronically relapsing disorder that persists following long periods of abstinence, suggesting that the underlying molecular changes are stable and endure for long periods even in the absence of drug. Here, we investigated Transforming Growth Factor-Beta Type I receptor (TGF-β R1) expression in the nucleus accumbens (NAc) following periods of withdrawal from cocaine self-administration (SA) and a sensitizing regimen of non-contingent cocaine. Rats were exposed to either (i) repeated systemic injections (cocaine or saline), or (ii) self-administration (cocaine or saline) and underwent a period of forced abstinence (either 1 or 7 days of drug cessation). Withdrawal from cocaine self-administration resulted in an increase in TGF-β R1 protein expression in the NAc compared to saline controls. This increase was specific for volitional cocaine intake as no change in expression was observed following a sensitizing regimen of experimenter-administered cocaine. These findings implicate TGF-β signaling as a novel potential therapeutic target for treating drug addiction.     

A proteomic study of Xanthomonas oryzae pv. oryzae in rice xylem sap

Xanthomonas oryzae pv. oryzae (Xoo) is the second most important rice pathogen, causing a disease called bacterial leaf blight. Xoo colonizes and infects the vascular tissue resulting in tissue necrosis and wilting causing significant yield losses worldwide. In this study Xoo infected vascular fluid (xylem sap) was recovered and analyzed for secreted Xoo proteins. Three independent experiments resulted in the identification of 324 different proteins, 64 proteins were found in all three samples which included many of the known virulence-associated factors. In addition, 10 genes encoding for the identified proteins were inactivated and one mutant displayed statistically a significant loss in virulence when compared to the wild type Xoo, suggesting that a new virulence-associated factor has been revealed. The usefulness of this approach in understanding the lifestyle and unraveling the virulence-associated factors of phytopathogenic vascular bacteria is discussed.     

Variation in Aboveground Cover Influences Soil Nitrogen Availability at Fine Spatial Scales Following Severe Fire in Subalpine Conifer Forests

Following fire, fine-scale variation in early successional vegetation and soil nutrients may influence development of ecosystem structure and function. We studied conifer forests burned by stand-replacing wildfire in Greater Yellowstone (Wyoming, USA) to address two questions: (1) How do the variability and spatial structure of aboveground cover and soil nitrogen availability change during the first 4 years following stand-replacing fire? (2) At fine scales (2–20 m), are postfire soil inorganic N pools and fluxes related to aboveground cover? Aboveground cover, soil N pools, and annual net N transformations were measured from 2001 to 2004 using a spatially explicit sampling design in four 0.25-ha plots that burned during summer 2000. Within-stand variability (coefficient of variation) in postfire live vegetative cover declined with time since fire, whereas variability in bare mineral soil, charred litter and fresh litter was greatest 2-3 years postfire. The soil nitrate pool was more variable than the soil ammonium pool, but annual net nitrification was less variable than annual net N mineralization. Spatial structure (quantified by semivariograms) was observed in some aboveground cover variables (for example, graminoids and fresh litter), but there was little spatial structure in soil N variables and no obvious congruence in spatial scales of autocorrelation for soil N and aboveground cover. Significant Spearman correlations (at the sample point) indicated that aboveground cover and soil N were coupled following severe fire, and the dominant influence was from aboveground cover to soil N, rather than from soil N to vegetation. Initial patterns of fire severity and re-vegetation contributed to fine-scale heterogeneity in soil N availability for at least 4 years after severe fire.     

Parthenolide sensitizes cells to X-ray-induced cell killing through inhibition of NF-kappaB and split-dose repair

Human cancers have multiple alterations in cell signaling pathways that promote resistance to cytotoxic therapy such as X rays. Parthenolide is a sesquiterpene lactone that has been shown to inhibit several pro-survival cell signaling pathways, induce apoptosis, and enhance chemotherapy-induced cell killing. We investigated whether parthenolide would enhance X-ray-induced cell killing in radiation resistant, NF-kappaB-activated CGL1 cells. Treatment with 5 microM parthenolide for 48 to 72 h inhibited constitutive NF-kappaB binding and cell growth, reduced plating efficiency, and induced apoptosis through stabilization of p53 (TP53), induction of the pro-apoptosis protein BAX, and phosphorylation of BID. Parthenolide also enhanced radiation-induced cell killing, increasing the X-ray sensitivity of CGL1 cells by a dose modification factor of 1.6. Flow cytometry revealed that parthenolide reduced the percentage of X-ray-resistant S-phase cells due to induction of p21 waf1/cip1 (CDKN1A) and the onset of G1/S and G2/M blocks, but depletion of radioresistant S-phase cells does not explain the observed X-ray sensitization. Further studies demonstrated that the enhancement of X-ray-induced cell killing by parthenolide is due to inhibition of split-dose repair.     

Neuromechanics: an integrative approach for understanding motor control

Neuromechanics seeks to understand how muscles, sense organs,motor pattern generators, and brain interact to produce coordinatedmovement, not only in complex terrain but also when confrontedwith unexpected perturbations. Applications of neuromechanicsinclude ameliorating human health problems (including prosthesisdesign and restoration of movement following brain or spinalcord injury), as well as the design, actuation and control ofmobile robots. In animals, coordinated movement emerges fromthe interplay among descending output from the central nervoussystem, sensory input from body and environment, muscle dynamics,and the emergent dynamics of the whole animal. The inevitablecoupling between neural information processing and the emergentmechanical behavior of animals is a central theme of neuromechanics.Fundamentally, motor control involves a series of transformationsof information, from brain and spinal cord to muscles to body,and back to brain. The control problem revolves around the specifictransfer functions that describe each transformation. The transferfunctions depend on the rules of organization and operationthat determine the dynamic behavior of each subsystem (i.e.,central processing, force generation, emergent dynamics, andsensory processing). In this review, we (1) consider the contributionsof muscles, (2) sensory processing, and (3) central networksto motor control, (4) provide examples to illustrate the interplayamong brain, muscles, sense organs and the environment in thecontrol of movement, and (5) describe advances in both roboticsand neuromechanics that have emerged from application of biologicalprinciples in robotic design. Taken together, these studiesdemonstrate that (1) intrinsic properties of muscle contributeto dynamic stability and control of movement, particularly immediatelyafter perturbations; (2) proprioceptive feedback reinforcesthese intrinsic self-stabilizing properties of muscle; (3) controlsystems must contend with inevitable time delays that can simplifyor complicate control; and (4) like most animals under a varietyof circumstances, some robots use a trial and error processto tune central feedforward control to emergent body dynamics.