Design and Validation of a Novel Generic Platform for the Production of Tetravalent IgG1-like Bispecific Antibodies.

We have designed and validated a novel generic platform for production of tetravalent IgG1-like chimeric bispecific Abs. The VH-CH1-hinge domains of mAb2 are fused through a peptidic linker to the N terminus of mAb1 H chain, and paired mutations at the CH1-CL interface mAb1 are introduced that force the correct pairing of the two different free L chains. Two different sets of these CH1-CL interface mutations, called CR3 and MUT4, were designed and tested, and prototypic bispecific Abs directed against CD5 and HLA-DR were produced (CD5xDR). Two different hinge sequences between mAb1 and mAb2 were also tested in the CD5xDR-CR3 or -MUT4 background, leading to bispecific Ab (BsAbs) with a more rigid or flexible structure. All four Abs produced bound with good specificity and affinity to CD5 and HLA-DR present either on the same target or on different cells. Indeed, the BsAbs were able to efficiently redirect killing of HLA-DR(+) leukemic cells by human CD5(+) cytokine-induced killer T cells. Finally, all BsAbs had a functional Fc, as shown by their capacity to activate human complement and NK cells and to mediate phagocytosis. CD5xDR-CR3 was chosen as the best format because it had overall the highest functional activity and was very stable in vitro in both neutral buffer and in serum. In vivo, CD5xDR-CR3 was shown to have significant therapeutic activity in a xenograft model of human leukemia.

Use of combined suspension laryngoscopy, flexible bronchoscopy and high frequency jet ventilation for Y-shaped airway stents delivery

La trachée et les bronches proximales sont de fins conduits subtils, ingénieusement structurés par une partie cartilagineuse antérieure résistante aux variations de pression et une partie membraneuse postérieure souple. Par leurs faibles volumes (espace mort) ils délivrent un grand pourcentage de l'air inspiré aux voies distales, puis au parenchyme pulmonaire, permettant les échanges de gaz. Cette belle harmonie respiratoire peut être rapidement mise à mal dès qu'un processus atteint ces voies respiratoires proximales, soit en les comprimant, processus sténosant, soit en affaiblissant leur structure, trachéo-bronchomalacie, soit en ouvrant leur paroi sur les structures médiastinales, fistule trachéo/broncho-médiastinales, pleurales ou autres. Le pronostic vital est alors rapidement engagé au vu de l'absolue nécessité du bon fonctionnement de ces fins conduits, une petite diminution du calibre de leurs fines lumières provoquant une baisse importante de leurs surfaces. Dans ces situations à haut potentiel de complication majeure les interventions endoscopiques pour restaurer l'intégrité de ces conduits sont alors fort risquées, et il est primordial de pouvoir les effectuer dans un cadre sécurisé au maximum. La réalisation de ces gestes par la technique décrite dans notre article « Use of combined suspension laryngoscopy, flexible bronchoscopy and high frequency jet ventilation forY-shaped airway stents delivery" permet la sécurité nécessaire à ces situations instable, en effet -la laryngoscopie en suspension expose les voies proximales en offrant un accès le plus large possible à l'arbre trachéobronchique ce qui permet l insertion de multiples instruments parfois volumineux, -la Jet ventilation assure une oxygénation et une ventilation adéquate par un fin cathéter placé soit dans le poumon sain, soit en distalité de la lésion -la bronchoscopie souple, passant au travers d'endroits exigus et courbes permet le déploiement sous vision direct, au millimètre près, de divers dispositifs. Cette association remplace avantageusement la technique traditionnelle qui insère les stents à l'aveugle, et en apnée, ce qui représente de haut risque de mauvais positionnement des stents avec des conséquences immédiates sur l'oxygénation et la ventilation souvent déjà bien altérées. Perspective et conclusion : cette technique est utile pour l'insertion des stents en Y, centraux, comme décrit dans notre article, et les indications peuvent être étendues aux stents distaux pour lesquels l'accès n'est parfois pas aisé avec le bronchoscope rigide, et pour d'autres interventions endoscopiques, laser, cryothérapie, radiofréquence ou l'insertion de nouveaux dispositifs.

Flexible social organization and high incidence of drifting in the sweat bee, Halictus scabiosae.

The very diverse social systems of sweat bees make them interesting models to study social evolution. Here we focus on the dispersal behaviour and social organization of Halictus scabiosae, a common yet poorly known species of Europe. By combining field observations and genetic data, we show that females have multiple reproductive strategies, which generates a large diversity in the social structure of nests. A detailed microsatellite analysis of 60 nests revealed that 55% of the nests contained the offspring of a single female, whereas the rest had more complex social structures, with three clear cases of multiple females reproducing in the same nest and frequent occurrence of unrelated individuals. Drifting among nests was surprisingly common, as 16% of the 122 nests in the overall sample and 44% of the nests with complex social structure contained females that had genotypes consistent with being full-sisters of females sampled in other nests of the population. Drifters originated from nests with an above-average productivity and were unrelated to their nestmates, suggesting that drifting might be a strategy to avoid competition among related females. The sex-specific comparison of genetic differentiation indicated that dispersal was male-biased, which would reinforce local resource competition among females. The pattern of genetic differentiation among populations was consistent with a dynamic process of patch colonization and extinction, as expected from the unstable, anthropogenic habitat of this species. Overall, our data show that H. scabiosae varies greatly in dispersal behaviour and social organization. The surprisingly high frequency of drifters echoes recent findings in wasps and bees, calling for further investigation of the adaptive basis of drifting in the social insects.

Flexible colony-founding strategies in a socially polymorphic ant

In social insects the number of queens per nest varies greatly. One of the proximate causes of this variation may be that queens produced by multiple-queen colonies are generally smaller, and might thus be unable to found new colonies independently. We examined whether the social origin of queens and males influenced the colony-founding success of queens in the socially polymorphic ant Formica selysi. Queens originating from single-queen and multiple-queen colonies had similar survival rates and colony-founding success, be they alone or in two-queen associations. During the first 5 months, queens originating from single-queen colonies gave rise to more workers than queens originating from multiple-queen colonies. Pairs of queens were also more productive than single queens. However, these differences in productivity were transient, as all types of colonies had reached a similar size after 15 months. Mating between social forms was possible and did not decrease queen survival or colony productivity, compared to mating within social forms. Overall, these results indicate that queens from each social form are able to found colonies independently, at least under laboratory conditions. Moreover, gene flow between social forms is not restricted by mating or genetic incompatibilities. This flexibility in mating and colony founding helps to explain the maintenance of alternative social structures in sympatry and the absence of genetic differentiation between social forms.

Structural macro factors and the affine term structure of interest rates

This work consists of three essays investigating the ability of structural macroeconomic models to price zero coupon U.S. government bonds. 1. A small scale 3 factor DSGE model implying constant term premium is able to provide reasonable a fit for the term structure only at the expense of the persistence parameters of the structural shocks. The test of the structural model against one that has constant but unrestricted prices of risk parameters shows that the exogenous prices of risk-model is only weakly preferred. We provide an MLE based variance-covariance matrix of the Metropolis Proposal Density that improves convergence speeds in MCMC chains. 2. Affine in observable macro-variables, prices of risk specification is excessively flexible and provides term-structure fit without significantly altering the structural parameters. The exogenous component of the SDF is separating the macro part of the model from the term structure and the good term structure fit has as a driving force an extremely volatile SDF and an implied average short rate that is inexplicable. We conclude that the no arbitrage restrictions do not suffice to temper the SDF, thus there is need for more restrictions. We introduce a penalty-function methodology that proves useful in showing that affine prices of risk specifications are able to reconcile stable macro-dynamics with good term structure fit and a plausible SDF. 3. The level factor is reproduced most importantly by the preference shock to which it is strongly and positively related but technology and monetary shocks, with negative loadings, are also contributing to its replication. The slope factor is only related to the monetary policy shocks and it is poorly explained. We find that there are gains in in- and out-of-sample forecast of consumption and inflation if term structure information is used in a time varying hybrid prices of risk setting. In-sample yield forecast are better in models with non-stationary shocks for the period 1982-1988. After this period, time varying market price of risk models provide better in-sample forecasts. For the period 2005-2008, out of sample forecast of consumption and inflation are better if term structure information is incorporated in the DSGE model but yields are better forecasted by a pure macro DSGE model.

Extending the canopy flow model for natural, highly flexible macrophyte canopies

Flow structures above vegetation canopies have received much attention within terrestrial and aquatic literature. This research has led to a good process understanding of mean and turbulent canopy flow structure. However, much of this research has focused on rigid or semi-rigid vegetation with relatively simple morphology. Aquatic macrophytes differ from this form, exhibiting more complex morphologies, predominantly horizontal posture in the flow and a different force balance. While some recent studies have investigated such canopies, there is still the need to examine the relevance and applicability of general canopy layer theory to these types of vegetation. Here, we report on a range of numerical experiments, using both semi-rigid and highly flexible canopies. The results for the semi-rigid canopies support existing canopy layer theory. However, for the highly flexible vegetation, the flow pattern is much more complex and suggests that a new canopy model may be required.

Structure-function relationships of the alpha1b-adrenergic receptor.

The alpha1b-adrenergic receptor (AR) is a member of the large superfamily of seven transmembrane domain (TMD) G protein-coupled receptors (GPCR). Combining site-directed mutagenesis of the alpha1b-AR with computational simulations of receptor dynamics, we have explored the conformational changes underlying the process of receptor activation, i.e. the transition between the inactive and active states. Our findings suggest that the structural constraint stabilizing the alpha1b-AR in the inactive form is a network of H-bonding interactions amongst conserved residues forming a polar pocket and R143 of the DRY sequence at the end of TMDIII. We have recently reported that point mutations of D142, of the DRY sequence and of A293 in the distal portion of the third intracellular loop resulted in ligand-independent (constitutive) activation of the alpha1b-AR. These constitutively activating mutations could induce perturbations resulting in the shift of R143 out of the polar pocket. The main role of R143 may be to mediate receptor activation by triggering the exposure of several basic amino acids of the intracellular loops towards the G protein. Our investigation has been extended also to the biochemical events involved in the desensitization process of alpha1b-AR. Our results indicate that immediately following agonist-induced activation, the alpha1b-AR can undergo rapid agonist-induced phosphorylation and desensitization. Different members of the G protein coupled receptor kinase family can play a role in agonist-induced regulation of the alpha1b-AR. In addition, constitutively active alpha1b-AR mutants display different phosphorylation and internalization features. The future goal is to further elucidate the molecular mechanism underlying the complex equilibrium between activation and inactivation of the alpha1b-AR and its regulation by pharmacological substances. These findings can help to elucidate the mechanism of action of various agents displaying properties of agonists or inverse agonists at the adrenergic system.

Kin structure and queen execution in the Argentine ant Linepithema humile

Every spring, workers of the Argentine Ant Linepithema humile kill a large proportion of queens within their nests, Although this behaviour inflicts a high energetic cost oil the colonies, its biological significance has remained elusive so far. An earlier study showed that the probability of a queen being executed is not related to her weight, fecundity, or age. Here we test the hypothesis that workers collectively eliminate queens to which they are less related, thereby increasing their inclusive fitness. We found no evidence for this hypothesis. Workers of a nest were on average not significantly less related to executed queens than to surviving ones. Moreover, a population genetic analysis revealed that workers were not genetically differentiated between nests. This means that workers of a given nest are equally related to any queen in the population and that there can be no increase in average worker-queen relatedness by selective elimination of queens. Finally, our genetic analyses also showed that, in contrast to workers, queens were significantly genetically differentiated between nests and that there was significant isolation by distance for queens.

Covariation between colony social structure and immune defences of workers in the ant Formica selysi

Several ant species vary in the number of queens per colony, yet the causes and consequences of this variation remain poorly understood. In previous experiments, we found that Formica selysi workers originating from multiple-queen (=polygyne) colonies had a lower resistance to a fungal pathogen than workers originating from single-queen (=monogyne) colonies. In contrast, group diversity improved disease resistance in experimental colonies. This discrepancy between field and experimental colonies suggested that variation in social structure in the field had antagonistic effects on worker resistance, possibly through a down-regulation of the immune system balancing the positive effect of genetic diversity. Here, we examined if workers originating from field colonies with alternative social structure differed in three major components of their immune system. We found that workers from polygyne colonies had a lower bacterial growth inhibitory activity than workers from monogyne colonies. In contrast, workers from the two types of colonies did not differ significantly in bacterial cell wall lytic activity and prophenoloxidase activity. Overall, the presence of multiple queens in a colony correlated with a slight reduction in one inducible component of the immune system of individual workers. This reduced level of immune defence might explain the lower resistance of workers originating from polygyne colonies despite the positive effect of genetic diversity. More generally, these results indicate that social changes at the group level can modulate individual immune defences.

Pizgrischite, (Cu,Fe)Cu14PbBi17S35, a new sulfosalt from the Swiss Alps: Description, crystal structure and occurrence

Pizgrischite, (Cu,Fe)Cu14PbBi17S35, is a new mineral species named after the type locality, Piz Grisch Mountain, Val Ferrera, Graubunden, Switzerland. This sulfosalt occurs as thin, striated, metallic lead-grey blades measuring up to I cm in length, embedded in quartz and associated with tetrahedrite, chalcopyrite, pyrite, sphalerite, emplectite and derivatives of the aikinite-bismuthinite series. In plane-polarized light, the new species is brownish grey with no perceptible pleochroism; under crossed nicols in oil immersion, it presents a weak anisotropy with dark brown tints. Minimum and maximum reflectance values (in %) in air are: 40.7-42.15 (470 nm), 41.2-43.1 (546 nm), 41.2-43.35 (589 nm) and 40.7-43.3 (650 nm). Cleavage is perfect along 001 I and well developed on {010}. Abundant polysynthetic twinning is observed on (010). The mean micro-indentation hardness is 190 kg/mm(2) (Mohs hardness 3.3), and the calculated density is 6.58 g/cm(3). Electron-microprobe analyses yield (wt%; mean result of seven analyses): Cu 16.48, Pb 2.10, Fe 0.77, Bi 60.70, Sb 0.35, S 19.16, Se 0.04, total 99.60. The resulting empirical chemical formula is (Cu15.24Fe0.80Pb0.60)(Sigma 16.64)(Bi17.07Sb0.17)(Sigma 17.24)(S35.09Se0.03)(Sigma 35.12), in accordance with the formula derived from the single-crystal refinement of the structure, (Cu,Fe)Cu14PbBi17S35. Pizgrischite is monoclinic, space group C2/m, with the following unit-cell parameters: a 35.054(2), b3.91123(I), c43.192(2) angstrom, beta 96.713(4)degrees, V5881.24 angstrom(3), Z=4. The strongest seven X-ray powder-diffraction lines [d in angstrom (I)(hkl)] are: 5.364(40)((6) over bar 04), 4.080(50)((8) over bar 05), 3.120(40)(118), 3.104(68)((3) over bar 18), 2.759(53) ((9) over bar 11),2.752(44)(910) and 1.956(100)(020). The crystal structure is an expanded monoclinic derivative of kupcikite. Pizgrischite belongs to the cuprobismutite series of bismuth sulfosalts but, sensu stricto, it is not a homologue of cuprobismutite. At the type locality. pizarischite is the result of the Alpine metamorphism under greenschist-facies conditions of pre-Tertiary hydrothermal Cu-Bi mineralization.

Structure du temps de travail des infirmières de santé publique: effets de l'âge des patients, de la nature des prestations fournies et du lieu du contact. [Structure of work times of public health nurses: effect of the age of patients, nature of care given and location of contact].

In the framework of health services research sponsored by the Swiss National Science Foundation, a research was undertaken of the activity of the large majority of the public health nurses working in the Swiss cantons of Vaud and Fribourg (total population 700,000). During one week, 130 nurses gathered, with a specially devised instrument, data on 4165 patient visits. Studying the duration of the contacts, one has distinguished contact duration per se (DC), duration of the travel time preceding the contact (DD), and total duration in relation with the contact (DTC-addition of the first two). It was noted that the three durations increased significantly with patient age (as regard travel time, this is explained by the higher proportion of home visits in higher age groups, as compared with visits at a health center). Examined according to location of the visit, contact duration per se (without travel) is higher for visits at home and in nursing homes than for those taking place at a health center. Looked at in respect to the care given (technical care, or basic nursing care, or both simultaneously), our data show that the provision of basic nursing care (alone or with technical care) doubles contact duration (from 20 to 42-45'). The analyses according to patient age shows that, at an advanced age (beyond 80 years particularly), there is an important increase of the visits where both types of care are given. However, contact duration per se shows a significant raise with age only for the group "technical care only"; it can be demonstrated that this is due to the fact that older patients require more complex technical acts (e.g., bladder care, as compared with simpler acts such as injection). A model of the relationships between patient age and contact duration is proposed: it is because of the increase in the proportions of home visits, of visits including basic nursing care, and of more complex technical acts that older persons require more of the working time of public health nurses.

Bacterial community structure of a pesticide-contaminated site and assessment of changes induced in community structure during bioremediation.

The introduction of culture-independent molecular screening techniques, especially based on 16S rRNA gene sequences, has allowed microbiologists to examine a facet of microbial diversity not necessarily reflected by the results of culturing studies. The bacterial community structure was studied for a pesticide-contaminated site that was subsequently remediated using an efficient degradative strain Arthrobacter protophormiae RKJ100. The efficiency of the bioremediation process was assessed by monitoring the depletion of the pollutant, and the effect of addition of an exogenous strain on the existing soil community structure was determined using molecular techniques. The 16S rRNA gene pool amplified from the soil metagenome was cloned and restriction fragment length polymorphism studies revealed 46 different phylotypes on the basis of similar banding patterns. Sequencing of representative clones of each phylotype showed that the community structure of the pesticide-contaminated soil was mainly constituted by Proteobacteria and Actinomycetes. Terminal restriction fragment length polymorphism analysis showed only nonsignificant changes in community structure during the process of bioremediation. Immobilized cells of strain RKJ100 enhanced pollutant degradation but seemed to have no detectable effects on the existing bacterial community structure.

The twist, writhe and overall shape of supercoiled DNA change during counterion-induced transition from a loosely to a tightly interwound superhelix. Possible implications for DNA structure in vivo.

A cryo-electron microscopy study of supercoiled DNA molecules freely suspended in cryo-vitrified buffer was combined with Monte Carlo simulations and gel electrophoretic analysis to investigate the role of intersegmental electrostatic repulsion in determining the shape of supercoiled DNA molecules. It is demonstrated here that a decrease of DNA-DNA repulsion by increasing concentrations of counterions causes a higher fraction of the linking number deficit to be partitioned into writhe. When counterions reach concentrations likely to be present under in vivo conditions, naturally supercoiled plasmids adopt a tightly interwound conformation. In these tightly supercoiled DNA molecules the opposing segments of interwound superhelix seem to directly contact each other. This form of supercoiling, where two DNA helices interact laterally, may represent an important functional state of DNA. In the particular case of supercoiled minicircles (178 bp) the delta Lk = -2 topoisomers undergo a sharp structural transition from almost planar circles in low salt buffers to strongly writhed "figure-eight" conformations in buffers containing neutralizing concentrations of counterions. Possible implications of this observed structural transition in DNA are discussed.