Hour‐by‐Hour Analysis for Increased Accuracy of Greenhouse Gas Emissions for a Low‐Energy Condominium Design

The seasonal and hourly variation of electricity grid emissions and building operational energy use are generally not accounted for in carbon footprint analyses of buildings. This work presents a technique for and results of such an analysis and quantifies the errors that can be encountered when these variations are not appropriately addressed. The study consists of an hour‐by‐hour analysis of the energy used by four different variations of a five‐story condominium building, with a gross floor area of approximately 9,290 square meters (m²), planned for construction in Markham, Ontario, Canada. The results of the case studied indicate that failure to account for variation can, for example, cause a 4% error in the carbon footprint of a building where ground source heat pumps are used and a 6% and 8% error in accounting for the carbon savings of wind and photovoltaic systems, respectively. After the building envelope was enhanced and sources of alternative energy were incorporated, the embodied greenhouse gas (GHG) emissions were more than 50% of the building's operational emissions. This work illustrates the importance of short‐time‐scale GHG analysis for buildings.     

Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana

Stevia rebaudiana leaves accumulate a mixture of at least eight different steviol glycosides. The pattern of glycosylation heavily influences the taste perception of these intensely sweet compounds. The majority of the glycosides are formed by four glucosylation reactions that start with steviol and end with rebaudioside A. The steps involve the addition of glucose to the C-13 hydroxyl of steviol, the transfer of glucose to the C-2' and C-3' of the 13-O-glucose and the addition of glucose to the hydroxyl of the C-4 carboxyl group. We used our collection of ESTs, an UDP-glucosyltransferase (UGT)-specific electronic probe and key word searches to identify candidate genes resident in our collection. Fifty-four expressed sequence tags (ESTs) belonging to 17 clusters were found using this procedure. We isolated full length cDNAs for 12 of the UGTs, cloned them into an expression vector, and produced recombinant enzymes in Escherichia coli. An in vitro glucosyltransferase activity enzyme assay was conducted using quercetin, kaempferol, steviol, steviolmonoside, steviolbioside, and stevioside as sugar acceptors, and (14)C-UDP-glucose as the donor. Thin layer chromatography was used to separate the products and three of the recombinant enzymes produced labelled products that co-migrated with known standards. HPLC and LC-ES/MS were then used to further define those reaction products. We determined that steviol UGTs behave in a regioselective manner and propose a modified pathway for the synthesis of rebaudioside A from steviol.     

Dual functions for WNT5A during cartilage development and in disease

Mouse and human genetic data suggests that Wnt5a is required for jaw development but the specific role in facial skeletogenesis is unknown. We mapped expression of WNT5A in the developing chicken skull and found that the highest expression was in early Meckel's cartilage but by stage 35 expression was decreased to background. We focused on chondrogenesis by targeting a retrovirus expressing WNT5A to the mandibular prominence prior to cell differentiation. Unexpectedly, there were no phenotypes in the first 6 days following injection; however later the mandibular bones and Meckel's cartilage were reduced or missing on the treated side. To examine the effects on cartilage differentiation we treated micromass cultures from mandibular mesenchyme with Wnt5a-conditioned media (CM). Similar to in vivo viral data, cartilage differentiates normally, but, after 6 days of culture, nearly all Alcian blue staining is lost. Collagen II and aggrecan were also decreased in treated cultures. The matrix loss was correlated with upregulation of metalloproteinases, MMP1, MMP13, and ADAMTS5 (codes for Aggrecanase). Moreover, Marimastat, an MMP and Aggrecanase inhibitor rescued cartilage matrix in Wnt5a-CM treated cultures. The pathways mediating these cartilage and RNA changes were investigated using luciferase assays. Wnt5a-CM was a potent inhibitor of the canonical pathway and strongly activated JNK/PCP signaling. To determine whether the matrix loss is mediated by repression of canonical signaling or activation of the JNK pathway we treated mandibular cultures with either DKK1, an antagonist of the canonical pathway, or a small molecule that antagonizes JNK signaling (TCS JNK 6o). DKK1 slightly increased cartilage formation and therefore suggested that the endogenous canonical signaling represses chondrogenesis. To test this further we added an excess of Wnt3a-CM and found that far fewer cartilage nodules differentiated. Since DKK1 did not mimic the effects of Wnt5a we excluded the canonical pathway from mediating the matrix loss phenotype. The JNK antagonist partially rescued the Wnt5a phenotype supporting this non-canonical pathway as the main mediator of the cartilage matrix degradation. Our study reveals two new roles for WNT5A in development and disease: 1) to repress canonical Wnt signaling in cartilage blastema in order to promote normal differentiation and 2) in conditions of excess to stimulate degradation of mature cartilage matrix via non-canonical pathways.     

Defining Sequence Space and Reaction Products within the Cyanuric Acid Hydrolase (AtzD)/Barbiturase Protein Family

Cyanuric acid hydrolases (AtzD) and barbiturases are homologous, found almost exclusively in bacteria, and comprise a rare protein family with no discernible linkage to other protein families or an X-ray structural class. There has been confusion in the literature and in genome projects regarding the reaction products, the assignment of individual sequences as either cyanuric acid hydrolases or barbiturases, and spurious connection of this family to another protein family. The present study has addressed those issues. First, the published enzyme reaction products of cyanuric acid hydrolase are incorrectly identified as biuret and carbon dioxide. The current study employed (13)C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to show that cyanuric acid hydrolase releases carboxybiuret, which spontaneously decarboxylates to biuret. This is significant because it revealed that homologous cyanuric acid hydrolases and barbiturases catalyze completely analogous reactions. Second, enzymes that had been annotated incorrectly in genome projects have been reassigned here by bioinformatics, gene cloning, and protein characterization studies. Third, the AtzD/barbiturase family has previously been suggested to consist of members of the amidohydrolase superfamily, a large class of metallohydrolases. Bioinformatics and the lack of bound metals both argue against a connection to the amidohydrolase superfamily. Lastly, steady-state kinetic measurements and observations of protein stability suggested that the AtzD/barbiturase family might be an undistinguished protein family that has undergone some resurgence with the recent introduction of industrial s-triazine compounds such as atrazine and melamine into the environment.     

Simian immunodeficiency virus engrafted with human immunodeficiency virus type 1 (HIV-1)-specific epitopes: replication, neutralization, and survey of HIV-1-positive plasma

To date, only a small number of anti-human immunodeficiency virus type 1 (HIV-1) monoclonal antibodies (MAbs) with relatively broad neutralizing activity have been isolated from infected individuals. Adequate techniques for defining how frequently antibodies of these specificities arise in HIV-infected people have been lacking, although it is generally assumed that such antibodies are rare. In order to create an epitope-specific neutralization assay, we introduced well-characterized HIV-1 epitopes into the heterologous context of simian immunodeficiency virus (SIV). Specifically, epitope recognition sequences for the 2F5, 4E10, and 447-52D anti-HIV-1 neutralizing monoclonal antibodies were introduced into the corresponding regions of SIVmac239 by site-directed mutagenesis. Variants with 2F5 or 4E10 recognition sequences in gp41 retained replication competence and were used for neutralization assays. The parental SIVmac239 and the neutralization-sensitive SIVmac316 were not neutralized by the 2F5 and 4E10 MAbs, nor were they neutralized significantly by any of the 96 HIV-1-positive human plasma samples that were tested. The SIV239-2F5 and SIV239-4E10 variants were specifically neutralized by the 2F5 and 4E10 MAbs, respectively, at concentrations within the range of what has been reported previously for HIV-1 primary isolates (J. M. Binley et al., J. Virol. 78:13232-13252, 2004). The SIV239-2F5 and SIV239-4E10 epitope-engrafted variants were used as biological screens for the presence of neutralizing activity of these specificities. None of the 92 HIV-1-positive human plasma samples that were tested exhibited significant neutralization of SIV239-2F5. One plasma sample exhibited >90% neutralization of SIV239-4E10, but this activity was not competed by a 4E10 target peptide and was not present in concentrated immunoglobulin G (IgG) or IgA fractions. We thus confirm by direct analysis that neutralizing activities of the 2F5 and 4E10 specificities are either rare among HIV-1-positive individuals or, if present, represent only a very small fraction of the total neutralizing activity in any given plasma sample. We further conclude that the structures of gp41 from SIVmac239 and HIV-1 are sufficiently similar such that epitopes engrafted into SIVmac239 can be readily recognized by the cognate anti-HIV-1 monoclonal antibodies.     

Equal inhibition of HIV replication by stereoisomers of phosphatidyl-azidothymidine. Lack of stereospecificity of lysosomal phospholipase A1.

Glycerol-1-P and glycerol-3-P stereoisomers of dipalmitoylphosphatidylazidothymidine were synthesized and found to have equal antiretroviral activity in HIV-infected HT4-6C cells. It was anticipated that the glycerol-1-P isomer would be less active because of slow metabolic conversion by cellular phospholipases A and C, but the antiretroviral results suggested that the human cell line (HT4-6C) may have phospholipases capable of hydrolyzing 2,3-dipalmitoyl-sn-glycerol-1-phospho-5'-azidothymidine (AZT). To evaluate this possibility, we purified lysosomal phospholipase A1, an enzyme known to play a major role in cellular phospholipid catabolism. This enzyme rapidly hydrolyzed both the sn-1 and sn-3 isomers of dipalmitoylphosphatidyl-AZT. We synthesized sn-2,3-dipalmitoyl-glycero-1-phosphocholine and found that it is also hydrolyzed readily by lysosomal phospholipase A1 although the Vmax, 59 mumol mg-1 h-1, is slightly lower than that of the sn-1,2-dipalmitoyl-glycero-3-phosphocholine, 89 mumol mg-1 h-1. In conclusion, our studies show that sn-2,3-dipalmitoyl-glycerol-1-phospho-AZT is equal in antiviral activity to sn-1,2-dipalmitoyl-glycero-3-phospho-AZT in HIV-infected HT4-6C cells. This surprising result is due in part to the lack of stereospecificity of lysosomal phospholipase A1.     

Pseudomonas vaccine: A phase I evaluation for cancer research

Summary A heptavalent lipopolysaccharide vaccine of Pseudomonas aeruginosa (Pseudogen) was administered at four dose levels (0.1, 0.25, 0.5, and 0.75 mg/m²) to patients with advanced metastatic cancer that had proved refractory to chemotherapy. The vaccine was administered subcutaneously twice weekly. Local toxicity was seen in erythema, edema, pain and tenderness at the site of injection, and painful regional lymphadenopathy; manifestations of systemic toxicity included fever, chills, myalgias, nausea, and vomiting. Toxicity showed a clear-cut dose dependence. The maximally tolerated dose by this route and schedule was 0.5 mg/m². A significant rise of antibody titers was observed at all four dose levels. Evaluation of the delayed cutaneous hypersensitivity response to recall antigens and to the pseudomonas vaccine, and quantification of peripheral blood T and B cell levels and of in vitro lymphocyte blastogenic responses to commonly used mitogens and pseudomonas vaccine failed to demonstrate significant change from pretreatment values. Clinical trials to evaluate the therapeutic efficacy of pseudomonas vaccine with or without chemotherapy can be undertaken safely with this route and schedule.     

Development of a tissue array for primary melanoma with long-term follow-up: discovering melanoma cell adhesion molecule as an important prognostic marker

Refining current prognostic capability is essential for improving the management of melanoma. This study was undertaken to develop a tumor array for the rapid assessment of novel prognostic markers in a series of specimens from melanoma patients with 7- to 10-year follow-up. A melanoma database of 120 patients with archival specimens was created after histopathological review of original specimens. A tissue array was developed allowing 480 biopsy samples from the 120 primary melanoma specimens to be embedded into a single paraffin block. This was sectioned and stained for the adhesion marker melanoma cell adhesion molecule (MCAM); after further review, 76 of the 120 specimens were suitable for further analysis. The slides were assessed by two independent observers without previous knowledge of the clinical outcome for staining positivity and stain intensity. Assessment of association between MCAM and clinicopathological features was carried out using chi-squared analysis, and univariate and Cox multivariate analyses were performed on the data. There was a high correlation between MCAM intensity and both Clark's level and Breslow thickness (Spearman correlation p < 0.001 for both). The data revealed that MACM was a highly specific prognostic marker for survival in univariate analysis (chi2 = 18, p < 0.0001). Subgroup analysis by stratification of the staining intensity revealed a sequentially worsening survival with increasing staining intensity (chi2 = 22.33, p < 0.0001). Multivariate analysis of survival showed MCAM to be an independent prognostic marker more accurate than all other clinicopathological parameters (p < 0.0001), including the Breslow depth. Further analysis within only intermediate-thickness tumors showed MCAM intensity added further refinement to outcome prediction (chi2 = 22.33, p < 0.0001). The tissue array provided a rapid method of analyzing up to 480 specimens within a single paraffin block. This will benefit many areas of plastic surgery research. The identification of adhesion markers revealed a valuable prognostic marker for predicting outcome and a potential target for therapeutic manipulation.     

A new origin for the maxillary jaw

One conserved feature of craniofacial development is that the first pharyngeal arch has two components, the maxillary and mandibular, which then form the upper and lower jaws, respectively. However, until now, there have been no tests of whether the maxillary cells originate entirely within the first pharyngeal arch or whether they originate in a separate condensation, cranial to the first arch. We therefore constructed a fate map of the pharyngeal arches and environs with a series of dye injections into stage 13-17 chicken embryos. We found that from the earliest stage examined, the major contribution to the maxillary bud is from post-optic mesenchyme with a relatively minor contribution from the maxillo-mandibular cleft. Cells labeled within the first pharyngeal arch contributed exclusively to the mandibular prominence. Gene expression data showed that there were different molecular codes for the cranial and caudal maxillary prominence. Two of the genes examined, Rarbeta (retinoic acid receptor beta) and Bmp4 (bone morphogenetic protein) were expressed in the post-optic mesenchyme and epithelium prior to formation of the maxillary prominence and then were restricted to the cranial half of the maxillary prominence. In order to determine the derivatives of the maxillary prominence, we performed focal injections of CM-DiI into the stage 24 maxillary prominence. Labeled cells contributed to the maxillary, palatine, and jugal bones, but not the other elements of the upper beak, the premaxilla and prenasal cartilage. We also determined that the cranial cells give rise to more distal parts of the upper beak, whereas caudal cells form proximal structures. Grafts of stage 24 maxillary prominences were also analyzed to determine skeletal derivatives and these results concurred with the DiI maps. These early and later fate maps indicate that the maxillary prominence and its skeletal derivatives are not derived from the first pharyngeal arch but rather from a separate maxillary condensation that occurs between the eye and the maxillo-mandibular cleft. These data also suggest that during evolution, recession of the first pharyngeal arch-derived palatoquadrate cartilage to a more proximal position gave way to the bony upper jaw of amniotes.