Environmental benchmarks for buildings: needs,challenges and solutions—71st LCA forum,Swiss Federal Institute of Technology,Zürich, 18 June 2019

The International Journal of Life Cycle Assessment - The 71st LCA forum was held on 18 June 2019 in Zurich, Switzerland, to discuss the current status and future plans of environmental benchmarking...     

Recombinant renewable polyclonal antibodies

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.     

Two step capture and purification of IgG2 using multicolumn countercurrent solvent gradient purification (MCSGP)

A two‐step chromatography process for monoclonal antibody (mAb) purification from clarified cell culture supernatant (cCCS) was developed using cation exchange Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) as a capture step. After an initial characterization of the cell culture supernatant the capture step was designed from a batch gradient elution chromatogram. A variety of chromatographic materials was screened for polishing of the MCSGP‐captured material in batch mode. Using multi‐modal anion exchange in bind‐elute mode, mAb was produced consistently within the purity specification. The benchmark was a state‐of‐the‐art 3‐step chromatographic process based on protein A, anion and cation exchange stationary phases. The performance of the developed 2‐step process was compared to this process in terms of purity, yield, productivity and buffer consumption. Finally, the potential of the MCSGP process was investigated by comparing its performance to that of a classical batch process that used the same stationary phase. Biotechnol. Bioeng. 2010;107: 974–984. © 2010 Wiley Periodicals, Inc.     

Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression,but non-specifically enhanced on induction of Tax expression

Background

Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4⁺ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis.

Results

Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ_26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02.

Conclusions

HTLV-1 gene expression in primary CD4⁺ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.

     

Parameterizing a model of Douglas fir water flow using a tracheid-level model

The theory of tree water flow proposed in Aumann & Ford (submitted) is assessed by numerically solving the model developed from this theory under a variety of functional parameterizations. The unknown functions in this nonlinear partial differential equation model are determined using a tracheid-level model of water flow in a block of Douglas fir tracheids. The processes of flow, cavitation, pit aspiration/deaspiration, flow through the cell wall and ray exudation in a block of approximately 79 000 tracheids are modeled. Output from the tracheid model facilitates determination of the hydraulic conductivities in the sapwood as a function of saturation and interfacial area between liquid and gaseous phases of water, the function governing the rate of change in saturation, and the function governing the rate of change in interfacial area. The models show complementary things. The tracheid model shows that capacitance, or the change in saturation per change in pressure, is not constant. When all refilling is stopped, it takes over 180 days for the hydraulic conductivity in the vertical direction to reach 1/4 of its maximal value, showing the robustness of the transpiration stream for conducting water. The shape of the functions determined with the tracheid model change with different tracheid-level assumptions. When these functions are used in the differential equation model, it is shown that cell-wall conductivity plays an important part in the lag in flow observed in many conifers. The flow velocities and rates of change in saturation predicted by the differential equation model agree with those measured in Douglas fir. Both models support the theory of tree water flow presented in Aumann & Ford (submitted) and undermine the theory that water flow in trees is analogous to the flow of current in electric circuits.     

The putative glutathione peroxidase gene of Plasmodium falciparum codes for a thioredoxin peroxidase

A putative glutathione peroxidase gene (Swiss-Prot accession number Z 68200) of Plasmodium falciparum, the causative agent of tropical malaria, was expressed in Escherichia coli and purified to electrophoretic homogeneity. Like phospholipid hydroperoxide glutathione peroxidase of mammals, it proved to be monomeric. It was active with H(2)O(2) and organic hydroperoxides but, unlike phospholipid hydroperoxide glutathione peroxidase, not with phosphatidylcholine hydroperoxide. With glutathione peroxidases it shares the ping-pong mechanism with infinite V(max) and K(m) when analyzed with GSH as substrate. As a homologue with selenocysteine replaced by cysteine, its reactions with hydroperoxides and GSH are 3 orders of magnitude slower than those of the selenoperoxidases. Unexpectedly, the plasmodial enzyme proved to react faster with thioredoxins than with GSH and most efficiently with thioredoxin of P. falciparum (Swiss-Prot accession number 202664). It is therefore reclassified as thioredoxin peroxidase. With plasmodial thioredoxin, the enzyme also displays ping-pong kinetics, yet with a limiting K(m) of 10 microm and a k(1)' of 0.55 s(-)1. The apparent k(1)' for oxidation with cumene, t-butyl, and hydrogen peroxides are 2.0 x 10(4) m(-1) s(-1), 3.3 x 10(3) m(-1) s(-1), and 2.5 x 10(3) m (-1) s(-1), respectively. k(2)' for reduction by autologous thioredoxin is 5.4 x 10(4) m(-1) s(-1) (21.2 m(-1) s(-1) for GSH). The newly discovered enzymatic function of the plasmodial gene product suggests a reconsideration of its presumed role in parasitic antioxidant defense.     

Low temperature X-ray investigation of structural distributions in myoglobin

The results of X-ray structure analysis of metmyoglobin at 300 K, 185 K, 165 K, 115 K and 80 K are reported. The lattice vectorsa andb decrease linearly with temperature whilec shows non-linearity above 180 K, indicating some type of phase transition. Cooling does change the myoglobin structure but only within the structural distribution as determined by individual x ² at room temperature. Two residues showed significant alternative positions for sidechains at higher temperatures while only one position is occupied at low temperatures. In the case of LEU 61 a jump between different positions of the side-chain reduces the potential barrier for the entrance of the O₂ molecule to the heme pocket.The mean square displacements, x ², of the individual residues decrease linearly with temperature in most cases, indicating a parabolic envelope for the potential responsible for motions. A separation of rotational and translational disorder of the entire molecule is discussed. Comparison with Mössbauer spectroscopy indicates that protein dynamics on a time scale faster than 10^-7 s is not simply a harmonic process. Extrapolation of the structural distributions toT=0 K shows that a large zero point distribution of the myoglobin structure exists, thus proving that there is no absolute energy minimum for one well defined conformation.Dedicated to Prof. H. Frauenfelder on his 65^th birthday