P301L tau expression affects glutamate release and clearance in the hippocampal trisynaptic pathway

Individuals at risk of developing Alzheimer's disease (AD) often exhibit hippocampal hyperexcitability. A growing body of evidence suggests that perturbations in the glutamatergic tripartite synapse may underlie this hyperexcitability. Here, we used a tau mouse model of AD (rTg(TauP301L)4510) to examine the effects of tau pathology on hippocampal glutamate regulation. We found a 40% increase in hippocampal vesicular glutamate transporter, which packages glutamate into vesicles, and has previously been shown to influence glutamate release, and a 40% decrease in hippocampal glutamate transporter 1, the major glutamate transporter responsible for removing glutamate from the extracellular space. To determine whether these alterations affected glutamate regulation in vivo, we measured tonic glutamate levels, potassium‐evoked glutamate release, and glutamate uptake/clearance in the dentate gyrus, cornu ammonis 3(CA3), and cornu ammonis 1(CA1) regions of the hippocampus. P301L tau expression resulted in a 4‐ and 7‐fold increase in potassium‐evoked glutamate release in the dentate gyrus and CA3, respectively, and significantly decreased glutamate clearance in all three regions. Both release and clearance correlated with memory performance in the hippocampal‐dependent Barnes maze task. Alterations in mice expressing P301L were observed at a time when tau pathology was subtle and before readily detectable neuron loss. These data suggest novel mechanisms by which tau may mediate hyperexcitability.

     

Contrasting effects of GPS device and harness attachment on adult survival of Lesser Black‐backed Gulls Larus fuscus and Great Skuas Stercorarius skua

Telemetry has become an important method for studying the biology and ecology of animals. However, the impact of tracking devices and their method of attachment on different species across multiple temporal scales has seldom been assessed. We compared the behavioural and demographic responses of two species of seabird, Lesser Black‐backed Gull Larus fuscus and Great Skua Stercorarius skua, to a GPS device attached using a crossover wing harness. We used telemetry information and monitoring of breeding colonies to compare birds equipped with a device and harness, and control birds without an attachment. We assessed whether tagged birds have lower short‐term breeding productivity or lower longer term overwinter return rates (indicative of overwinter survival) than controls. For Great Skua, we also assessed whether territory attendance within the breeding season differed between tagged and control birds. As with previous studies on Lesser Black‐backed Gull, we found no short‐term impacts on breeding productivity or long‐term impacts on overwinter return rates. For Great Skua, there was no evidence for impacts of the device and harness on territory attendance or breeding productivity. However, as found by a previous study of Great Skuas using a different (body) harness design, there was strong evidence of reduced overwinter return rates. Consequently, a device attached using a wing harness was considered suitable for long‐term deployment on Lesser Black‐backed Gulls, but not on Great Skuas. These findings will inform the planning of future tracking studies.     

Using Common Boundaries to Assess Methane Emissions: A Life Cycle Evaluation of Natural Gas and Coal Power Systems

There is consensus on the importance of upstream methane (CH₄) emissions to the life cycle greenhouse gas (GHG) footprint of natural gas systems, but inconsistencies among recent studies explain why some researchers calculate a CH₄ emission rate of less than 1% whereas others calculate a CH₄ emission rate as high as 10%. These inconsistencies arise from differences in data collection methods, data collection time frames, and system boundaries. This analysis focuses on system boundary inconsistencies. Our results show that the calculated CH₄ emission rate can increase nearly fourfold not by changing the magnitude of any particular emission source, but by merely changing the portions of the supply chain that are included within the system boundary. Our calculated CH₄ emission rate for extraction through pipeline transmission is 1.2% for current practices. Our model allows us to identify GHG contributors in the upstream supply chain, but also allows us to tie upstream findings to complete life cycle scenarios. If applied to the life cycles of power systems and assessed in terms of cumulative radiative forcing, the upstream CH₄ emission rate can be as high as 3.2% before the GHG impacts from natural gas power exceed those from coal power at any point during a 100‐year time frame.     

Vascular Function in Grape Berries across Development and Its Relevance to Apparent Hydraulic Isolation

During the latter stages of development in fleshy fruit, water flow through the xylem declines markedly and the requirements of transpiration and further expansion are fulfilled primarily by the phloem. We evaluated the hypothesis that cessation of water transport through the xylem results from disruption or occlusion of pedicel and berry xylem conduits (hydraulic isolation). Xylem hydraulic resistance (R_h) was measured in developing fruit of grape (Vitis vinifera ‘Chardonnay’) 20 to 100 d after anthesis (DAA) and compared with observations of xylem anatomy by light and cryo-scanning electron microscopy and expression of six plasma membrane intrinsic protein (PIP) aquaporin genes (VvPIP1;1, VvPIP1;2, VvPIP1;3, VvPIP2;1, VvPIP2;2, VvPIP2;3). There was a significant increase in whole berry R_h and receptacle R_h in the latter stages of ripening (80–100 DAA), which was associated with deposition of gels or solutes in many receptacle xylem conduits. Peaks in the expression of some aquaporin isoforms corresponded to lower whole berry R_h 60 to 80 DAA, and the increase in R_h beginning at 80 DAA correlated with decreases in the expression of the two most predominantly expressed PIP genes. Although significant, the increase in berry R_h was not great enough, and occurred too late in development, to explain the decline in xylem flow that occurs at 60 to 75 DAA. The evidence suggests that the fruit is not hydraulically isolated from the parent plant by xylem occlusion but, rather, is “hydraulically buffered” by water delivered via the phloem.The development of grape (Vitis vinifera) berries is typical of many fleshy fruits, following a double sigmoid pattern of growth with three distinct phases: an initial phase of rapid cell division and expansion in green berries, a short transitory phase of very little growth, and a final phase in which growth is reinitiated and the fruit ripens. The transition to the ripening phase is accompanied by many physiological changes, such as the production of anthocyanins and fruit softening. In grape, these distinctive and highly visible physiological changes are collectively referred to as veraison. The rapid accumulation of sugars that is initiated in the berry mesocarp around the time of veraison is accompanied by a dramatic shift in the proportion of xylem and phloem transport (Lang and Thorpe, 1989; Greenspan et al., 1994, 1996). This same shift, albeit more gradual, occurs in many other fleshy fruits such as tomato (Solanum lycopersicum; Ho et al., 1987), apple (Malus domestica; Lang and Ryan, 1994; Drazeta et al., 2004), and kiwifruit (Actinidia deliciosa; Dichio et al., 2003) as well as in the flowers of tropical trees (Chapotin et al., 2003). The sudden reduction in xylem transport to the fruit is perceived as a mechanism to hydraulically isolate the fruit and buffer them from environmental stresses experienced by the parent plant.Using mass balance techniques, Greenspan et al. (1994, 1996) reported major changes in the role of the xylem and phloem in water transport to the grape berry at veraison. During the first growth phase, the xylem provides the majority of water transport into the berry. In the final growth stage, the phloem provides more than 80% of the berry''s water requirements and the contribution of the xylem becomes negligible. Berry water status also becomes apparently uncoupled from plant water status after veraison. Before veraison, diurnal contractions in berry diameter were strongly related to changes in plant (stem) water potential, while after veraison, diurnal contractions were greatly reduced and unrelated to changes in stem water potential (Matthews and Shackel, 2005). A similar lack of response was also observed for mesocarp cell turgor after veraison (Thomas et al., 2006). Thus, it is clear that some mechanism acts to decouple berry water relations from the water status of the parent plant.Over the past two decades, a general consensus has developed that the berry xylem becomes physically disrupted after veraison, effectively blocking the xylem pathway and isolating the fruit essentially as a whole from the parent plant (During et al., 1987; Findlay et al., 1987; Lang and Ryan, 1994). Evidence for this has been provided by observations of dye uptake into the berry through the xylem. When the cut pedicel of a preveraison berry is submerged in dye, the dye is taken up into peripheral and axial xylem conduits of the entire berry (Findlay et al., 1987; Creasy et al., 1993; Rogiers et al., 2001). After veraison, dye uptake is limited to the base of the berry vasculature (brush). From this evidence, together with micrographs that appeared to show stretched and ruptured xylem conduits in postveraison berries, it was inferred that the lignified tracheids present at veraison were physically torn apart by the expansion of the berry that occurred postveraison.Recent experimental work using a range of techniques suggests that the hypothesis of physical disruption may be oversimplified and that the berry xylem remains at least potentially functional after veraison (Bondada et al., 2005; Keller et al., 2006; Chatelet et al., 2008b). The results of Chatelet et al. (2008a, 2008b) demonstrate that the majority of xylem conduits in the berry remain intact after veraison and suggest that xylem development (growth of new conduits) continues well into the postveraison growth phase. Using both a modified pressure plate/membrane apparatus and a wicking technique, it was demonstrated that dye moved through the xylem of postveraison berries when a hydrostatic pressure or matric gradient was applied between the pedicel and the cut stylar surface (Bondada et al., 2005; Chatelet et al., 2008b). Keller et al. (2006) demonstrated this in reverse, showing that berry xylem was still capable of conducting a dye tracer back to the parent plant if the dye was introduced at the cut stylar end while the plant was transpiring. Thus, given a large enough pressure gradient, the xylem of postveraison berries retains the potential to transport water between the parent plant and the berry or vice versa. However, anatomical measurements and dye tracer studies can only be used to infer the degree to which fruit may become isolated from the parent plant. Knowledge of changes in hydraulic resistance (R_h) is required to determine whether xylem dysfunction is actually responsible for declining xylem flows reported with the progression of ripening. It is also important to differentiate between xylem flows and R_h, as these variables are sometimes confused in the literature; xylem flow rates can vary independently of R_h if water potential gradients along the pathway are altered.Previous studies examining changes in R_h associated with the development of fleshy fruit generally indicate that R_h increases during ripening but show differences in the timing and location of the increase. Some fruits develop an abscission zone in the pedicel or receptacle that is associated with vascular constriction and high R_h (Mackenzie, 1988; Lee, 1989; Van Ieperen et al., 2003). However, although some table grapes are believed to develop an abscission zone, there is no evidence of an abscission zone in wine grapes (Pratt, 1971). Tyerman et al. (2004) reported a substantial increase in R_h of grape berries after veraison, although this increase in resistance did not occur in the pedicel or receptacle but mainly in the distal section of the berries. Similarly, Malone and Andrews (2001) evaluated R_h in developing tomato fruits and stems and found that R_h increased in the fruit, but not proximal to the calyx. In apple, Lang and Ryan (1994) observed an increase in R_h at 80 d after anthesis (DAA) and also reported an increasing proportion of samples in which the xylem was completely occluded with age. Although they described these data as pedicel R_h, their measurements actually included the fruit vascular pathway; therefore, it is difficult to determine if the increase in R_h was manifested in the fruit or the pedicel.Increases in pedicel and receptacle R_h should be associated with changes in the dimensions or conductive state or xylem conduits. An increase in the R_h within the fruit may relate either to xylem dysfunction or to extravascular resistance beyond the xylem. Although they were not able to partition an increase in fruit R_h between the apoplast and symplast, Tyerman et al. (2004) suggested that the site of increased resistance may be the plasma membranes of vascular parenchyma cells separating xylem conduits and mesocarp cells rather than the xylem itself. A likely candidate driving hydraulic isolation at the cellular level is changes in plasma membrane R_h resulting from the differential expression and activity of aquaporins. Aquaporins are a family of transmembrane proteins considered to be largely responsible for the high permeability to water exhibited by plasma membranes. The regulation of R_h by aquaporins is now well documented in roots (Martre et al., 2001; McElrone et al., 2007; Vandeleur et al., 2009), and oxidative gating of aquaporins has been reported to reduce hydraulic conductivity by 90% in cells of the giant algae Chara (Henzler et al., 2004). The results of previous work suggest that aquaporins play an important role in the regulation of water movement during the development of flowers, seeds, and fruits (Maurel et al., 1995; Gao et al., 1999; Picaud et al., 2003; Shiota et al., 2006; Zhou et al., 2007). Changes in the expression of the plasma membrane intrinsic protein (PIP) PIP1 and PIP2 aquaporin gene families have been noted in ripening grapes (Picaud et al., 2003; Fei et al., 2004), although the effects of these changes on water transport (membrane conductivity) have not been documented. An increase of R_h between the mesocarp cells and the xylem within the fruit could provide a mechanism to restrict water movement between the parent plant and the berry if a large gradient in xylem tension existed between the two (Tyerman et al., 2004).While the concept of hydraulic isolation is generally accepted as part of the physiology of fleshy fruit development, we note that no studies have demonstrated an increase in R_h that is coincident with the decline in xylem flow. Additionally, measured variation in the R_h of the fruit and pedicel has not been quantitatively related to the water requirements of the fruit, taking into account water potential gradients between the fruit and the parent plant. In this study, we examined changes in the R_h of the berry, receptacle, and pedicel of Chardonnay grape over the course of fruit development. These measurements were compared against observations of xylem anatomy and aquaporin gene expression in order to investigate the hypotheses that (1) occlusion and/or disruption of xylem conduits results in the hydraulic isolation of ripening grape berries, and (2) an increase in the R_h of the berry is associated with changes in the expression of aquaporin genes in the mesocarp.     

Pooled-Peptide Epitope Mapping Strategies Are Efficient and Highly Sensitive: An Evaluation of Methods for Identifying Human T Cell Epitope Specificities in Large-Scale HIV Vaccine Efficacy Trials

The interferon gamma, enzyme-linked immunospot (IFN-γ ELISpot) assay is widely used to identify viral antigen-specific T cells is frequently employed to quantify T cell responses in HIV vaccine studies. It can be used to define T cell epitope specificities using panels of peptide antigens, but with sample and cost constraints there is a critical need to improve the efficiency of epitope mapping for large and variable pathogens. We evaluated two epitope mapping strategies, based on group testing, for their ability to identify vaccine-induced T-cells from participants in the Step HIV-1 vaccine efficacy trial, and compared the findings to an approach of assaying each peptide individually. The group testing strategies reduced the number of assays required by >7-fold without significantly altering the accuracy of T-cell breadth estimates. Assays of small pools containing 7–30 peptides were highly sensitive and effective at detecting single positive peptides as well as summating responses to multiple peptides. Also, assays with a single 15-mer peptide, containing an identified epitope, did not always elicit a response providing validation that 15-mer peptides are not optimal antigens for detecting CD8+ T cells. Our findings further validate pooling-based epitope mapping strategies, which are critical for characterizing vaccine-induced T-cell responses and more broadly for informing iterative vaccine design. We also show ways to improve their application with computational peptide:MHC binding predictors that can accurately identify the optimal epitope within a 15-mer peptide and within a pool of 15-mer peptides.     

Robust Classification of Small-Molecule Mechanism of Action Using a Minimalist High-Content Microscopy Screen and Multidimensional Phenotypic Trajectory Analysis

Phenotypic screening through high-content automated microscopy is a powerful tool for evaluating the mechanism of action of candidate therapeutics. Despite more than a decade of development, however, high content assays have yielded mixed results, identifying robust phenotypes in only a small subset of compound classes. This has led to a combinatorial explosion of assay techniques, analyzing cellular phenotypes across dozens of assays with hundreds of measurements. Here, using a minimalist three-stain assay and only 23 basic cellular measurements, we developed an analytical approach that leverages informative dimensions extracted by linear discriminant analysis to evaluate similarity between the phenotypic trajectories of different compounds in response to a range of doses. This method enabled us to visualize biologically-interpretable phenotypic tracks populated by compounds of similar mechanism of action, cluster compounds according to phenotypic similarity, and classify novel compounds by comparing them to phenotypically active exemplars. Hierarchical clustering applied to 154 compounds from over a dozen different mechanistic classes demonstrated tight agreement with published compound mechanism classification. Using 11 phenotypically active mechanism classes, classification was performed on all 154 compounds: 78% were correctly identified as belonging to one of the 11 exemplar classes or to a different unspecified class, with accuracy increasing to 89% when less phenotypically active compounds were excluded. Importantly, several apparent clustering and classification failures, including rigosertib and 5-fluoro-2’-deoxycytidine, instead revealed more complex mechanisms or off-target effects verified by more recent publications. These results show that a simple, easily replicated, minimalist high-content assay can reveal subtle variations in the cellular phenotype induced by compounds and can correctly predict mechanism of action, as long as the appropriate analytical tools are used.     

Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis

A key component of a sound functional genomics infrastructure is the availability of a knockout mutant for every gene in the genome. A fruitful approach to systematically knockingout genes in the plant Arabidopsis thaliana has been the use of transferred-DNA (T-DNA) from Agrobacterium tumefaciens as an insertional mutagen. One of the assumptions underlying the use of T-DNA as a mutagen is that the insertion of these DNA elements into the Arabidopsis genome occurs at randomly selected locations. We have directly investigated the distribution of T-DNA insertions sites in populations of transformed Arabidopsis using two different approaches. To begin with, we utilized a polymerase chain reaction (PCR) procedure to systematically catalog the precise locations of all the T-DNA elements inserted within a 65 kb segment of chromosome IV. Of the 47 T-DNA insertions identified, 30% were found within the coding regions of genes. We also documented the insertion of T-DNA elements within the centromeric region of chromosome IV. In addition to these targeted T-DNA screens, we also mapped the genomic locations of 583 randomly chosen T-DNA elements by sequencing the genomic DNA flanking the insertion sites from individual T-DNA-transformed lines. 35% of these randomly chosen T-DNA insertions were located within the coding regions of genes. For comparison, coding sequences account for 44% of the Arabidopsis genome. Our results demonstrate that there is a small bias towards recovering T-DNA insertions within intergenic regions. However, this bias does not limit the utility of T-DNA as an effective insertional mutagen for use in reverse-genetic strategies.     

Rat epidermal keratinocytes as an organotypic model for examining the role of Cx43 and Cx26 in skin differentiation

In order to characterize connexin expression and regulation in the epidermis, we have characterized a rat epidermal keratinocyte (REK) cell line that is phenotypically similar to basal keratinocytes in that they have the ability to differentiate into organotypic epidermis consisting of a basal cell layer, 2-3 suprabasal cell layers, and a cornified layer. RT-PCR revealed that REK cells express mRNA for Cx26, Cx31, Cx31.1, Cx37, and Cx43, which mimics the reported connexin profile for rat tissue. In addition, we report the expression of Cx30, Cx30.3, Cx40, and Cx45 in rat keratinocytes, highlighting the complexity of the connexin complement in rat epidermis. Furthermore, 3-dimensional analysis of organotypic skin revealed that Cx26 and Cx43 are exquisitely regulated during the differentiation process. The life-cycle of these connexins including their expression, transport, assembly into gap junctions, internalization, and degradation are elegantly depicted in organotypic epidermis as keratinocytes proceed from differentiation to programmed cell death.     

Lymphocyte senescence in COPD is associated with loss of glucocorticoid receptor expression by pro-inflammatory/cytotoxic lymphocytes

Background

Glucocorticoid (GC) resistance is a major barrier in COPD treatment. We have shown increased expression of the drug efflux pump, Pgp1 in cytotoxic/pro-inflammatory lymphocytes in COPD. Loss of lymphocyte co-stimulatory molecule CD28 (lymphocyte senescence) was associated with a further increase in their pro-inflammatory/cytotoxic potential and resistance to GC. We hypothesized that lymphocyte senescence and increased Pgp1 are also associated with down-regulation of the GC receptor (GCR).

Methods

Blood was collected from 10 COPD and 10 healthy aged-matched controls. Flow cytometry was applied to assess intracellular pro-inflammatory cytokines, CD28, Pgp1, GCR, steroid binding and relative cytoplasm/nuclear GCR by CD28+ and CD28null T, NKT-like cells. GCR localization was confirmed by fluorescent microscopy.

Results

COPD was associated with increased numbers of CD28nullCD8+ T and NKT-like cells. Loss of CD28 was associated with an increased percentage of T and NKT-like cells producing IFNγ or TNFα and associated with a loss of GCR and Dex-Fluor staining but unchanged Pgp1. There was a significant loss of GCR in CD8 + CD28null compared with CD8 + CD28+ T and NKT-like cells from both COPD and controls (eg, mean ± SEM 8 ± 3% GCR + CD8 + CD28null T-cells vs 49 ± 5% GCR + CD8 + CD28+ T-cells in COPD). There was a significant negative correlation between GCR expression and IFNγ and TNFα production by T and NKT-like cells(eg, COPD: T-cell IFNγ R = −.615; ) and with FEV1 in COPD (R = −.777).

Conclusions

COPD is associated with loss of GCR in senescent CD28null and NKT-like cells suggesting alternative treatment options to GC are required to inhibit these pro-inflammatory/cytotoxic cells.     

Regulation of ion transport by pH and [HCO3-] in isolated gills of the crab Neohelice (Chasmagnathus) granulata

Posterior isolated gills of Neohelice (Chasmagnathus) granulatus were symmetrically perfused with hemolymph-like saline of varying [HCO3-] and pH. Elevating [HCO3-] in the saline from 2.5 to 12.5 mmol/l (pH 7.75 in both cases) induced a significant increase in the transepithelial potential difference (Vte), a measure of ion transport. The elevation in [HCO3-] also induced a switch from acid secretion (-43.7 +/- 22.5 microequiv.kg(-1).h(-1)) in controls to base secretion (84.7 +/- 14.4 microequiv.kg(-1).h(-1)). The HCO3(-)-induced Vte increase was inhibited by basolateral acetazolamide (200 micromol/l), amiloride (1 mmol/l), and ouabain (5 mmol/l) but not by bafilomycin (100 nmol/l). The Vte response to HCO3(-) did not take place in Cl(-)-free conditions; however, it was unaffected by apical SITS (2 mmol/l) or DIDS (1 mmol/l). A decrease in pH from 7.75 to 7.45 pH units in the perfusate also induced a significant increase in Vte, which was matched by a net increase in acid secretion of 67.8 +/- 18.4 microequiv kg(-1) h(-1). This stimulation was sensitive to basolateral acetazolamide, bafilomycin, DIDS, and Na+-free conditions, but it still took place in Cl(-)-free saline. Therefore, the cellular response to low pH is different from the HCO3(-)-stimulated response. We also report V-H+-ATPase- and Na+-K+-ATPase-like immunoreactivity in gill sections for the first time in this crab. Our results suggest that carbonic anhydrase (CA), basolateral Na+/H+ exchangers and Na+-K+-ATPase and apical anion exchangers participate in the HCO3(-)-stimulated response, while CA, apical V-H+-ATPase and basolateral HCO3(-)-dependent cotransporters mediate the response to low pH.