Emission of hydrogen sulfide (H2S) at a waterfall in a sewer: study of main factors affecting H2S emission and modeling approaches.

Hydrogen sulfide (H2S) represents one of the main odorant gases emitted from sewer networks. A mathematical model can be a fast and low-cost tool for estimating its emission. This study investigates two approaches to modeling H2S gas transfer at a waterfall in a discharge manhole. The first approach is based on an adaptation of oxygen models for H2S emission at a waterfall and the second consists of a new model. An experimental set-up and a statistical data analysis allowed the main factors affecting H2S emission to be studied. A new model of the emission kinetics was developed using linear regression and taking into account H2S liquid concentration, waterfall height and fluid velocity at the outlet pipe of a rising main. Its prediction interval was estimated by the residual standard deviation (15.6%) up to a rate of 2.3 g H2S·h-1. Finally, data coming from four sampling campaigns on sewer networks were used to perform simulations and compare predictions of all developed models.

Construction of Synthetic Antibody Libraries.

Libraries of antibody fragments displayed on filamentous phages are now a widely used approach to isolate antibodies against virtually any target. We describe a simple protocol to make large and diverse libraries based on a single or a limited number of frameworks. The approach is flexible enough to be used with any antibody format, either single-chain (scFv, VHH) or multi-chain (Fv, Fab, (Fab')2), and to target in a single step the six complementarity-determining regions-or any other part-of the antibody molecule. Using this protocol, libraries larger than 1010 can be constructed in a single week.

Construction of a Synthetic Antibody Gene Library for the Selection of Intrabodies and Antibodies.

Libraries of antibody fragments displayed on filamentous phages have proved their value to generate human antibodies against virtually any target. We describe here a simple protocol to make large and diverse libraries based on a single or a limited number of frameworks. The approach is flexible enough to be used with any antibody format, either single-chain (scFv, VHH) or multi-chain (Fv, Fab, (Fab')2), and to target in a single step the six complementarity-determining regions-or any other part-of the antibody molecule. Using this protocol, libraries larger than 1010 can be easily constructed in a single week.

In-cell intrabody selection from a diverse human library identifies C12orf4 protein as a new player in rodent mast cell degranulation.

The high specificity of antibodies for their antigen allows a fine discrimination of target conformations and post-translational modifications, making antibodies the first choice tool to interrogate the proteome. We describe here an approach based on a large-scale intracellular expression and selection of antibody fragments in eukaryotic cells, so-called intrabodies, and the subsequent identification of their natural target within living cell. Starting from a phenotypic trait, this integrated system allows the identification of new therapeutic targets together with their companion inhibitory intrabody. We applied this system in a model of allergy and inflammation. We first cloned a large and highly diverse intrabody library both in a plasmid and a retroviral eukaryotic expression vector. After transfection in the RBL-2H3 rat basophilic leukemia cell line, we performed seven rounds of selection to isolate cells displaying a defect in FcεRI-induced degranulation. We used high throughput sequencing to identify intrabody sequences enriched during the course of selection. Only one intrabody was common to both plasmid and retroviral selections, and was used to capture and identify its target from cell extracts. Mass spectrometry analysis identified protein RGD1311164 (C12orf4), with no previously described function. Our data demonstrate that RGD1311164 is a cytoplasmic protein implicated in the early signaling events following FcεRI-induced cell activation. This work illustrates the strength of the intrabody-based in-cell selection, which allowed the identification of a new player in mast cell activation together with its specific inhibitor intrabody.