Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering

By combining the knowledge gained from an analysis of the biophysical properties of natural antibody variable domains, the effects of mutations obtained in directed evolution experiments, and the detailed structural comparison of antibodies, it has now become possible to engineer antibodies for higher thermodynamic stability and more efficient folding. This is particularly important when antibodies are to be used under conditions where the disulfide bonds cannot form, i.e., in intracellular applications (as "intrabodies"). We describe in detail two methods for the knowledge-based improvement of antibody stability and folding efficiency. While CDR grafting from a non-human to the most closely related human antibody framework is an established technique to reduce the immunogenicity of a therapeutic antibody, CDR grafting for stabilization implies the use of a more distantly related acceptor framework with superior biophysical characteristics. The use of such dissimilar frameworks requires particular attention to antigen contact residues outside the classical CDR definition and to residues capable of indirectly affecting the conformation of the antigen binding site. As a second alternative, the stability of a suboptimal framework can be improved by the introduction of point mutations designed to optimize key residue interactions. We describe the analysis methods used to identify such point mutations, which can be introduced all at once, while maintaining the framework features necessary for antigen binding. These rational approaches render the continued "rediscovery" of certain mutations by directed evolution unnecessary, but they can also be used in conjunction with such methods to discover even better molecules.     

Synthesis and characterization of degradable multivalent cationic lipids with disulfide-bond spacers for gene delivery

Gene therapy provides powerful new approaches to curing a large variety of diseases, which are being explored in ongoing worldwide clinical trials. To overcome the limitations of viral gene delivery systems, synthetic nonviral vectors such as cationic liposomes (CLs) are desirable. However, improvements of their efficiency at reduced toxicity and a better understanding of their mechanism of action are required. We present the efficient synthesis of a series of degradable multivalent cationic lipids (CMVLn, n=2 to 5) containing a disulfide bond spacer between headgroup and lipophilic tails. This spacer is designed to be cleaved in the reducing milieu of the cytoplasm and thus decrease lipid toxicity. Small angle X-ray scattering demonstrates that the initially formed lamellar phase of CMVLn-DNA complexes completely disappears when reducing agents such as DTT or the biologically relevant reducing peptide glutathione are added to mimic the intracellular milieu. The CMVLs (n=3 to 5) exhibit reduced cytotoxicity and transfect mammalian cells with efficiencies comparable to those of highly efficient non-degradable analogs and benchmark commercial reagents such as Lipofectamine 2000. Thus, our results demonstrate that degradable disulfide spacers may be used to reduce the cytotoxicity of synthetic nonviral gene delivery carriers without compromising their transfection efficiency.     

Enumeration of Bacteriophages and Host Bacteria in Sewage and the Activated-Sludge Treatment Process

Bacteriophage populations in an activated-sludge sewage treatment plant were enumerated. A newly developed assay for quantitation of total phages, employing direct electron microscopic counts, was used in conjunction with the plaque assay. The total concentration of phages was significantly higher in reactor mixed liquor and effluent than in influent sewage, indicating a net production of phages within the reactor. Maximum total phage concentrations in the fluid phase of sewage, activated-sludge mixed liquor, and reactor effluent were 2.2 × 10⁷, 9.5 × 10⁷, and 8.4 × 10⁷/ml, respectively. Conditions were optimized for isolation of predominant heterotrophic aerobic bacteria from sewage and mixed liquor. Blending at ice water temperatures was superior to ultrasound or enzyme treatments for maximum release of viable bacteria from microbial floc. A solidified extract of mixed liquor was superior to standard media for cultivating maximum numbers of heterotrophic bacteria. The highest culture counts for sewage and mixed liquor were 1.4 × 10⁷ and 1.3 × 10⁹/ml, respectively, which represented only 3 and 6.8% of the total microscopic cell counts. Only 3 out of 48 dominant bacterial isolates from either mixed liquor or sewage were hosts for phages present in the system. The sum of phage populations infecting these three hosts accounted for, at best, 3.8% (sewage) and 0.2% (mixed liquor) of the total number of phages present. Generally, specific phage titers were lower in mixed liquor than in sewage, indicating that these hosts were not responsible for the net production of phages in the reactor. This study emphasizes the limitations of the plaque assay for ecological studies of phages, and it suggests that bacteria responsible for phage production in activated-sludge mixed liquor are either minor components of the heterotrophic population, floc-producing strains, or members of other physiological groups.     

Nest site choice compensates for climate effects on sex ratios in a lizard with environmental sex determination

Theoretical models suggest that in changing environments natural selection on two traits, maternal nesting behaviour and pivotal temperatures (those that divide the sexes) is important for maintaining viable offspring sex ratios in species with environmental sex determination (ESD). Empirical evidence, however, is lacking. In this paper, we provide such evidence from a study of clinal variation in four sex-determining traits (maternal nesting behaviour, pivotal temperatures, nesting phenology, and nest depth) in Physignathus lesueurii, a wide-ranging ESD lizard inhabiting eastern Australia. Despite marked differences in air and soil temperatures across our five study sites spanning 19° latitude and 1200 m in elevation, nest temperatures did not differ significantly among sites. Lizards compensated for climatic differences chiefly by selecting more open nest sites with higher incident radiation at cooler sites. Clinal variation in the onset of nesting also compensated for climatic differences, but to a lesser extent. There was no evidence of compensation through pivotal temperatures or nest depth. More broadly, our results extend to the egg stage the life history prediction that behaviour is the chief compensatory mechanism for climatic differences experienced by species spanning environmental extremes. Furthermore, our study was unique in revealing that nest site choice influenced mainly the daily range in nest temperatures, rather than mean temperatures, in a shallow-nesting reptile. Finally, indirect evidence suggests that the cue used by nesting lizards was radiation or temperature (through basking or assessing substrate temperatures), not visual detection of canopy openness. We conclude that maternal nesting behaviour and nesting phenology are traits subject to sex ratio selection in P. lesueurii, and thus, must be considered among the repertoire of ESD species for responding to climate change.     

The implication of irrigation in climate change impact assessment: a European‐wide study

This study evaluates the impacts of projected climate change on irrigation requirements and yields of six crops (winter wheat, winter barley, rapeseed, grain maize, potato, and sugar beet) in Europe. Furthermore, the uncertainty deriving from consideration of irrigation, CO₂ effects on crop growth and transpiration, and different climate change scenarios in climate change impact assessments is quantified. Net irrigation requirement (NIR) and yields of the six crops were simulated for a baseline (1982–2006) and three SRES scenarios (B1, B2 and A1B, 2040–2064) under rainfed and irrigated conditions, using a process‐based crop model, SIMPLACE . We found that projected climate change decreased NIR of the three winter crops in northern Europe (up to 81 mm), but increased NIR of all the six crops in the Mediterranean regions (up to 182 mm yr^?1). Climate change increased yields of the three winter crops and sugar beet in middle and northern regions (up to 36%), but decreased their yields in Mediterranean countries (up to 81%). Consideration of CO₂ effects can alter the direction of change in NIR for irrigated crops in the south and of yields for C3 crops in central and northern Europe. Constraining the model to rainfed conditions for spring crops led to a negative bias in simulating climate change impacts on yields (up to 44%), which was proportional to the irrigation ratio of the simulation unit. Impacts on NIR and yields were generally consistent across the three SRES scenarios for the majority of regions in Europe. We conclude that due to the magnitude of irrigation and CO₂ effects, they should both be considered in the simulation of climate change impacts on crop production and water availability, particularly for crops and regions with a high proportion of irrigated crop area.     

On-Chip Imaging of Schistosoma haematobium Eggs in Urine for Diagnosis by Computer Vision

Background

Microscopy, being relatively easy to perform at low cost, is the universal diagnostic method for detection of most globally important parasitic infections. As quality control is hard to maintain, misdiagnosis is common, which affects both estimates of parasite burdens and patient care. Novel techniques for high-resolution imaging and image transfer over data networks may offer solutions to these problems through provision of education, quality assurance and diagnostics. Imaging can be done directly on image sensor chips, a technique possible to exploit commercially for the development of inexpensive “mini-microscopes”. Images can be transferred for analysis both visually and by computer vision both at point-of-care and at remote locations.

Methods/Principal Findings

Here we describe imaging of helminth eggs using mini-microscopes constructed from webcams and mobile phone cameras. The results show that an inexpensive webcam, stripped off its optics to allow direct application of the test sample on the exposed surface of the sensor, yields images of Schistosoma haematobium eggs, which can be identified visually. Using a highly specific image pattern recognition algorithm, 4 out of 5 eggs observed visually could be identified.

Conclusions/Significance

As proof of concept we show that an inexpensive imaging device, such as a webcam, may be easily modified into a microscope, for the detection of helminth eggs based on on-chip imaging. Furthermore, algorithms for helminth egg detection by machine vision can be generated for automated diagnostics. The results can be exploited for constructing simple imaging devices for low-cost diagnostics of urogenital schistosomiasis and other neglected tropical infectious diseases.     

Labeled Trichoderma reesei Cellulase as a Marker for Acanthamoeba Cyst Wall Cellulose in Infected Tissues

Some protozoans are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent. Acanthamoeba is an exception since its cyst wall contains cellulose. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1,4-linked hexose backbones of these molecules. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. The identification of Acanthamoeba spp., which is based primarily on morphological and biochemical features, is labor-intensive and requires cloning and axenization. We describe a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of Trichoderma reesei cellulase to protozoan cyst wall cellulose. A recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from T. reesei cellulases was coupled to the fluorescent dyes Alexa Fluor 350 and Alexa Fluor 568 or was labeled with biotin using EZ-Link sulfo-NHS-biotin. No staining reaction was observed with chitin-containing preparations of fungi. Thus, the recombinant CBDs can be used as a marker to distinguish between cellulose and chitin. This allows rapid identification of Acanthamoeba cyst wall cellulose in paraffin or frozen sections of infected tissues.Laboratory diagnosis of infections with Acanthamoeba spp. is based on identification of the parasite in infected tissue. Although various techniques, including immunocytological and molecular methods, have been described, recovery of viable parasites by cultivation on agar is still the basic procedure used (16). This method is usually associated with histopathological examination of the specimen to prove tissue invasion by the parasite.Recognition of parasites in tissue sections is often difficult and depends on the expertise of the pathologist. In addition to traditional histological staining methods, immunohistology using parasite-specific antibodies, lectin conjugates, and calcofluor white have been used for visualization of parasites in tissue sections (3).Some protozoan parasites have the ability to protect themselves by forming a cyst wall, which is resistant to environmental stresses such as desiccation, lack of nutrients, and variations in temperature and pH. In most pathogenic protozoans studied, chitin is the carbohydrate polymer providing the required structural toughness to the cyst wall. Acanthamoeba spp. are exceptions, as their cysts are made up of cellulose. Recently, cellulose has also been identified as a cyst wall component in a closely related amoeba, Balamuthia mandrillaris (15). Cellulose consists of β-d-glucosyl units linked by β-1,4-glucosidic bonds. Chitin is very similar but contains N-acetylglucosamine as the monomer. Both polymers form very similar crystalline macroscopic structures. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1,4-linked hexose backbones. This is especially true for various fluorescent brightening agents, such as calcofluor white, used as cytochemical markers in microscopic diagnosis of protozoan and fungal infections. A two-domain structural organization is often observed in cellulose-degrading enzymes. Most Trichoderma reesei cellulases consist of a catalytic domain and a cellulose-binding domain (CBD) joined by a linker. The catalytic domain contains the active site with the amino acid residues responsible for the hydrolytic mechanism. The role of the CBD is to bind to the solid cellulose. The ability of CBDs to attach to cellulose can be utilized in various applications. Individual types of CBDs can vary significantly in their properties, such as affinity, preference for crystalline or amorphous cellulose, and cross-reactivity with other similar carbohydrates (7, 8, 9, 10).We have previously described a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of T. reesei cellulase to protozoan cyst wall cellulose (12). In that study we used a recombinant dimeric CBD (D-CBD) fusion protein in an indirect immunofluorescence analysis to specifically stain the cellulose and visualize its localization in the cyst wall. In preliminary studies, this method was also shown to be useful detection of parasites in tissue sections (11).The aim of the present study was to simplify the detection method by preparing D-CBDs as fluorescent and biotinylated conjugates that could be used for direct and rapid detection of cellulose in Acanthamoeba by both fluorescence and ordinary light microscopy.     

Functional attributes of two subtropical shrubs and implications for the distribution and management of endangered bird habitat

Aims The fruits of Erithalis fruticosa L. and Lantana involucrata L. are important in the diet of US federally endangered Kirtland's Warblers (Setophaga kirtlandii) wintering in the Bahamas archipelago. These two shrubs occur in tropical and subtropical dry forests, including forests that have been subjected to recent disturbance. Despite their importance to the endangered warbler, the disturbance ecology of these shrubs is poorly understood. We sought to determine, based on functional characteristics of the plants, whether their presence is favored by a particular type or regime of disturbance.Methods We used data from field experiments (seed broadcasting and shrub cutting) conducted on the island of Eleuthera, The Bahamas to determine mechanisms of and conditions favoring establishment and persistence ('vital attributes') of E. fruticosa and L. involucrata, which enabled categorization according to the plant functional types defined by Noble and Slatyer (1980). We then compared hypothesized distributions of these plant functional types among different anthropogenic disturbance regimes to observed distributions of E. fruticosa and L. involucrata in order to identify disturbance regimes most likely to produce habitat used by Kirtland's Warblers.Important findings E. fruticosa and L. involucrata were functionally categorized as widely dispersed but largely shade intolerant species capable of establishing or regenerating individuals after disturbance via both seeds and vegetative mechanisms. Both hypothesized and observed distribution patterns indicated the shrubs were favored by a regime of frequent disturbance producing open canopy and ground layers. Among the anthropogenic disturbances we examined, areas of large-scale land clearing combined with subsequent goat grazing most often supported E. fruticosa and L. involucrata, while the shrubs were relatively rare in burned areas. Utilizing the plant functional type framework in combination with field data to evaluate predictions of species occurrence among different disturbances regimes provides a strong theoretical basis for conservation strategies. Understanding which disturbance types favor a habitat of concern and the mechanisms by which they do so can aid the prioritization of areas for protection or the design of habitat management protocols.     

Pretecto-tectal influences

(1)From the dorsal surface of the toad (Bufo b. spinosus, B. marinus) optic tectum (OT), field potentials (FP) were recorded at 9 reference sites in response to electrical stimulation of the optic nerve (ON). The FP showed 4 main components, besides an initial deflection attributed to axonal potentials: two negative waves N1, N2 (attributed to postsynaptic excitatory processes) and two positive waves P2, P3 (attributed to postsynaptic inhibitory processes). The responses across the reference sites were rather similar in different individuals. (2) Electrical stimulation of an area in the ipsilateral pretectal lateral posterodorsal and posterior (Lpd/P) thalamic region evoked tectal FPs showing mainly a negative and a positive wave. Regarding wave amplitudes, the FPs displayed disproportionalities across the reference sites. (3) Electrical stimulation of the contralateral Lpd/P evoked mainly a positive wave in the tectal FP whose disproportionality corresponded roughly to the one obtained to ipsilateral Lpd/P stimulation. (4) The inital negative wave of the tectal FP in response to ON stimulation was nearly abolished, if Lpd/P stimulation preceded ON stimulation at a delay of 17–25 ms. (5) Since FPs showed adaptation to repetitive stimulation, various experiments were carried out to distinguish adaptation phenomena from effects of neuronal interactions between Lpd/P and OT. (6) The results provide evidence that ON- and Lpd/P-mediated inputs interact in superficial tectal layers, whereby pretectotectal input suppresses retinotectal excitatory information transfer. Input of Lpd/P to the contralateral superficial OT suggests postsynaptic inhibition. This study provides no information about pretectal inputs to deeper tectal layers, which anatomically are known to exist.Abbreviations A-I recording sites from the dorsal tectal surface - D _t delay between Lpd/P and ON stimulation - EPSP IPSP excitatory and inhibitory postsynaptic potentials, respectively - FP field potential - L latency of FP waves - ON optic nerve - OT optic tectum - Lpd/P lateral posterodorsal and posterior pretectal thalamic region - Lpv lateral posteroventral pretectal thalamic nucleus - N, P negative and positive waves of FPs, respectively - PRE presynaptic axonal input - TH pretectal thalamic neurons