Chimeric yellow fever/dengue virus as a candidate dengue vaccine: quantitation of the dengue virus-specific CD8 T-cell response

We have constructed a chimeric yellow fever/dengue (YF/DEN) virus, which expresses the premembrane (prM) and envelope (E) genes from DEN type 2 (DEN-2) virus in a YF virus (YFV-17D) genetic background. Immunization of BALB/c mice with this chimeric virus induced a CD8 T-cell response specific for the DEN-2 virus prM and E proteins. This response protected YF/DEN virus-immunized mice against lethal dengue encephalitis. Control mice immunized with the parental YFV-17D were not protected against DEN-2 virus challenge, indicating that protection was mediated by the DEN-2 virus prM- and E-specific immune responses. YF/DEN vaccine-primed CD8 T cells expanded and were efficiently recruited into the central nervous systems of DEN-2 virus challenged mice. At 5 days after challenge, 3 to 4% of CD8 T cells in the spleen were specific for the prM and E proteins, and 34% of CD8 T cells in the central nervous system recognized these proteins. Depletion of either CD4 or CD8 T cells, or both, strongly reduced the protective efficacy of the YF/DEN virus, stressing the key role of the antiviral T-cell response.     

Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO‐cell derived biotherapeutics

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non‐electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science‐based process validation strategies to ensure viral safety of biotechnology products. Biotechnol. Bioeng. 2009; 104: 371–380 © 2009 Wiley Periodicals, Inc.     

Comment on M. Guillot‐Delost et al. (2008;10:834‐846): Clinical‐grade preparation of human natural regulatory T cells encoding the thymidine kinase suicide gene as a safety gene

This Letter to the Editor has a published Response, which can be viewed using the link below. Response to Comment     

Spag17 Deficiency Results in Skeletal Malformations and Bone Abnormalities

Height is the result of many growth and development processes. Most of the genes associated with height are known to play a role in skeletal development. Single-nucleotide polymorphisms in the SPAG17 gene have been associated with human height. However, it is not clear how this gene influences linear growth. Here we show that a targeted mutation in Spag17 leads to skeletal malformations. Hind limb length in mutants was significantly shorter than in wild-type mice. Studies revealed differences in maturation of femur and tibia suggesting alterations in limb patterning. Morphometric studies showed increased bone formation evidenced by increased trabecular bone area and the ratio of bone area to total area, leading to reductions in the ratio of marrow area/total area in the femur. Micro-CTs and von Kossa staining demonstrated increased mineral in the femur. Moreover, osteocalcin and osterix were more highly expressed in mutant mice than in wild-type mice femurs. These data suggest that femur bone shortening may be due to premature ossification. On the other hand, tibias appear to be shorter due to a delay in cartilage and bone development. Morphometric studies showed reduction in growth plate and bone formation. These defects did not affect bone mineralization, although the volume of primary bone and levels of osteocalcin and osterix were higher. Other skeletal malformations were observed including fused sternebrae, reduced mineralization in the skull, medial and metacarpal phalanges. Primary cilia from chondrocytes, osteoblasts, and embryonic fibroblasts (MEFs) isolated from knockout mice were shorter and fewer cells had primary cilia in comparison to cells from wild-type mice. In addition, Spag17 knockdown in wild-type MEFs by Spag17 siRNA duplex reproduced the shorter primary cilia phenotype. Our findings disclosed unexpected functions for Spag17 in the regulation of skeletal growth and mineralization, perhaps because of its role in primary cilia of chondrocytes and osteoblasts.     

Neural Correlates of Emotional Interference in Social Anxiety Disorder

Disorder-relevant but task-unrelated stimuli impair cognitive performance in social anxiety disorder (SAD); however, time course and neural correlates of emotional interference are unknown. The present study investigated time course and neural basis of emotional interference in SAD using event-related functional magnetic resonance imaging (fMRI). Patients with SAD and healthy controls performed an emotional stroop task which allowed examining interference effects on the current and the succeeding trial. Reaction time data showed an emotional interference effect in the current trial, but not the succeeding trial, specifically in SAD. FMRI data showed greater activation in the left amygdala, bilateral insula, medial prefrontal cortex (mPFC), dorsal anterior cingulate cortex (ACC), and left opercular part of the inferior frontal gyrus during emotional interference of the current trial in SAD patients. Furthermore, we found a positive correlation between patients’ interference scores and activation in the mPFC, dorsal ACC and left angular/supramarginal gyrus. Taken together, results indicate a network of brain regions comprising amygdala, insula, mPFC, ACC, and areas strongly involved in language processing during the processing of task-unrelated threat in SAD. However, specifically the activation in mPFC, dorsal ACC, and left angular/supramarginal gyrus is associated with the strength of the interference effect, suggesting a cognitive network model of attentional bias in SAD. This probably comprises exceeded allocation of attentional resources to disorder-related information of the presented stimuli and increased self-referential and semantic processing of threat words in SAD.     

Tyloses and Phenolic Deposits in Xylem Vessels Impede Water Transport in Low-Lignin Transgenic Poplars: A Study by Cryo-Fluorescence Microscopy

Of 14 transgenic poplar genotypes (Populus tremula × Populus alba) with antisense 4-coumarate:coenzyme A ligase that were grown in the field for 2 years, five that had substantial lignin reductions also had greatly reduced xylem-specific conductivity compared with that of control trees and those transgenic events with small reductions in lignin. For the two events with the lowest xylem lignin contents (greater than 40% reduction), we used light microscopy methods and acid fuchsin dye ascent studies to clarify what caused their reduced transport efficiency. A novel protocol involving dye stabilization and cryo-fluorescence microscopy enabled us to visualize the dye at the cellular level and to identify water-conducting pathways in the xylem. Cryo-fixed branch segments were planed in the frozen state on a sliding cryo-microtome and observed with an epifluorescence microscope equipped with a cryo-stage. We could then distinguish clearly between phenolic-occluded vessels, conductive (stain-filled) vessels, and nonconductive (water- or gas-filled) vessels. Low-lignin trees contained areas of nonconductive, brown xylem with patches of collapsed cells and patches of noncollapsed cells filled with phenolics. In contrast, phenolics and nonconductive vessels were rarely observed in normal colored wood of the low-lignin events. The results of cryo-fluorescence light microscopy were supported by observations with a confocal microscope after freeze drying of cryo-planed samples. Moreover, after extraction of the phenolics, confocal microscopy revealed that many of the vessels in the nonconductive xylem were blocked with tyloses. We conclude that reduced transport efficiency of the transgenic low-lignin xylem was largely caused by blockages from tyloses and phenolic deposits within vessels rather than by xylem collapse.Secondary xylem in woody plants is a complex vascular tissue that functions in mechanical support, conduction, storage, and protection (Carlquist, 2001; Tyree and Zimmermann, 2002). The xylem must provide a sufficient and safe water supply throughout the entire pathway from roots to leaves for transpiration and photosynthesis. It is well established that enhanced water conductivity of xylem can increase total plant carbon gain (Domec and Gartner, 2003; Santiago et al., 2004; Brodribb and Holbrook, 2005a). According to the Hagen-Poiseuille equation, xylem conductivity should scale with vessel lumen diameter to the fourth power (Tyree and Zimmermann, 2002). Indeed, xylem conductivity largely depends on anatomical features, including conduit diameters and frequencies (Salleo et al., 1985; McCulloh and Sperry, 2005). However, there are hydraulic limits to maximum vessel diameters, because xylem conduits have to withstand the strong negative pressures of the transpiration stream that could cause cell collapse or embolisms within vessels that are structurally inadequate to withstand these forces (Tyree and Sperry, 1989; Lo Gullo et al., 1995; Hacke et al., 2000). To some extent, stomatal regulation of transpiration limits the negative pressures that the xylem experiences (Tardieu and Davies, 1993; Cochard et al., 2002; Meinzer, 2002; Brodribb and Holbrook, 2004; Buckley, 2005; Franks et al., 2007; Woodruff et al., 2007). Nevertheless, plants rely on an array of structural reinforcements of xylem to ensure the safety of water transport. The size of xylem elements, vessel redundancy, intervessel pit and membrane geometries, and the thickness, microstructure, and chemical composition of cell walls are among the features that regulate tradeoffs between efficiency and safety of xylem water transport (Baas and Schweingruber, 1987; Hacke et al., 2001; Domec et al., 2006; Ewers et al., 2007; Choat et al., 2008).The xylem cell wall is made up of cellulose bundles that are hydrogen bonded with hemicelluloses, which are in turn embedded within a lignin matrix (Mansfield, 2009; Salmén and Burgert, 2009). Besides providing this matrix for the cell wall itself, lignin is thought to contribute to many of the mechanical and physical characteristics of wood as well as conferring passive resistance to the spread of pathogens within a plant (Niklas, 1992; Boyce et al., 2004; Davin et al., 2008). Lignin typically represents 20% to 30% of the dry mass of wood and therefore is among the most abundant stores of carbon in the biosphere (Zobel and van Buijtenen, 1989). The complex molecular structure and biosynthetic pathway of various types of lignins have been studied extensively (Boerjan et al., 2003; Ralph et al., 2004, 2007; Higuchi, 2006; Boudet, 2007; Davin et al., 2008). The monomeric composition of lignin varies between different cell types of the same species depending on the functional specialization of the cell (Yoshinaga et al., 1992; Watanabe et al., 2004; Xu et al., 2006). The composition and amount of lignin in wild plants varies in response to climatic conditions (Donaldson, 2002) or gravitational and mechanical demands (Pruyn et al., 2000; Kern et al., 2005; Rüggeberg et al., 2008). It is clear that plants are capable of regulating the lignification pattern in differentiating cells, which provides them with flexibility for responding to environmental stresses (Donaldson, 2002; Koehler and Telewski, 2006; Ralph et al., 2007; for review, see Vanholme et al., 2008).Whereas some level of lignin is a requisite for all vascular plants, it is often an unwanted product in the pulp and paper industry because it increases the costs of paper production and associated water treatments necessary for environmental protection (Chen et al., 2001; Baucher et al., 2003; Peter et al., 2007). Reducing the lignin content of the raw biomass material may allow more efficient hydrolysis of polysaccharides in biomass and thus facilitate the production of biofuel (Chen and Dixon, 2007). With the ultimate goal of development of wood for more efficient processing, much research has been aimed at the production of genetically modified trees with altered lignin biosynthesis (Boerjan et al., 2003; Boudet et al., 2003; Li et al., 2003; Halpin, 2004; Ralph et al., 2004, 2008; Chiang, 2006; Coleman et al., 2008a, 2008b; Vanholme et al., 2008; Wagner et al., 2009). It is now technically possible to achieve more than 50% reductions of lignin content in xylem of poplar (Populus spp.; Leplé et al., 2007; Coleman et al., 2008a, 2008b), but the consequences of such reduction on plant function have received relatively little attention (Koehler and Telewski, 2006). In-depth studies on the xylem structure and functional performance of transgenic plants with low lignin are limited, despite the need to assess their long-term sustainability for large-scale production (Anterola and Lewis, 2002; Hancock et al., 2007; Coleman et al., 2008b, Voelker, 2009; Horvath et al., 2010).Genetically modified plants are suitable models for studying fundamental questions of the physiological role of lignin because of the possibility of controlling lignification without the confounding effects encountered when comparing across plant tissues or stages of development (Koehler and Telewski, 2006; Leplé et al., 2007; Coleman et al., 2008b). Research on Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) has shown that down-regulation of lignin biosynthesis can have diverse effects on plant metabolism and structure, including changes in the lignin amount and composition (p-hydroxyphenyl/guaiacyl/syringyl units ratio) as well as the collapse of xylem vessel elements (Lee et al., 1997; Sewalt et al., 1997; Piquemal et al., 1998; Chabannes et al., 2001; Jones et al., 2001; Franke et al., 2002; Dauwe et al., 2007). Among temperate hardwoods, poplar has been established as a model tree for genetic manipulations because of its ecological and economic importance, fast growth, ease of vegetative propagation, and its widespread use in traditional breeding programs (Bradshaw et al., 2001; Brunner et al., 2004). The question of how manipulation of lignin can affect the anatomy and physiological function of xylem in poplar has been addressed in part by several research groups (Anterola and Lewis, 2002; Boerjan et al., 2003; Leplé et al., 2007; Coleman et al., 2008b). Some studies that involved large lignin reductions reported no significant alterations in the xylem anatomy (Hu et al., 1999; Li et al., 2003). However, in many other experiments, reduced total lignin content was associated with significant growth retardation, alterations in the lignin monomer composition, irregularities in the xylem structure (Anterola and Lewis, 2002; Leplé et al., 2007; Coleman et al., 2008b), and the patchy occurrence of collapsed xylem cells (Coleman et al., 2008b; Voelker, 2009). Furthermore, severely down-regulated lignin biosynthesis has resulted in greatly reduced xylem water-transport efficiency (Coleman et al., 2008b; Lachenbruch et al., 2009; Voelker, 2009). It is generally assumed that the reduced water transport ability of xylem with very low lignin contents is caused by collapsed conduits and/or increased embolism due to the entry of air bubbles into the water-conducting cells (Coleman et al., 2008b; Wagner et al., 2009), but detailed anatomical investigations of the causes of impaired xylem conductivity of low-lignin trees are lacking. Analysis of the anatomical basis for the properties of xylem conduits in plants with genetically manipulated amounts and composition of lignin can provide a deeper understanding of the physiological role of lignin as well as the lower limit of down-regulation of lignin biosynthesis at which trees can still survive within natural environments.One of the approaches for the suppression of lignin biosynthesis is down-regulation of 4-coumarate:coenzyme A ligase (4CL), an enzyme that functions in phenylpropanoid metabolism by producing the monolignol precursor p-coumaroyl-CoA (Kajita et al.,1997; Allina et al., 1998; Hu et al., 1998; Harding et al., 2002; Jia et al., 2004; Costa et al., 2005; Friedmann et al., 2007; Wagner et al., 2009). In a 2-year-long field trial on the physiological performance of poplar (Populus tremula × Populus alba) transgenic clones, out of 14 genotypes with altered lignin biosynthesis (down-regulated 4CL), five showed dramatically reduced wood-specific conductivity (k_s) compared with that of control trees (Voelker, 2009). Those mutants with the severely reduced k_s were also characterized by having the lowest wood lignin contents (up to an approximately 40% reduction) in the study. Trees with transgenic events characterized by the formation of abnormally brown wood exhibited regular branch dieback at the end of the growing season, despite having been regularly watered (Voelker, 2009). Our objective was to identify the structural features responsible for reduced transport efficiency in the xylem of transgenic poplars with extremely low lignin contents. We employed fluorescence and laser scanning confocal microscopy for anatomical analyses of xylem structure as well as dye-flow experiments followed by cryo-fluorescence microscopy to visualize the functioning water-conductive pathways in xylem at the cellular level. We report the frequent occurrence of tyloses and phenolic depositions in xylem vessels of strongly down-regulated trees that may be the cause of their reduced xylem conductivity.     

Grand challenge commentary: Exploiting single-cell variation for new antibiotics

Variations between single members of a bacterial population can lead to antibiotic resistance that is not gene based. The future of effective infectious disease management might depend on a better understanding of this phenomenon and the potential to manipulate both it and microbial population dynamics in general.