Method for generation of human hyperdiversified antibody fragment library

The selection of antibody fragments from libraries using in vitro screening technologies has proven to be a very good alternative to the classical hybridoma technology, and has overcome the laborious process of antibody humanization. However, the complexity of the library is critical in the probability of being able to directly isolate a high affinity antibody specific to a target. We report a method to make hyperdiversified antibody fragment libraries, based on human immunoglobulin variable genes mimicking the somatic hypermutation process. This mutagenesis technology, MutaGen, was used for the first time on the entire variable domain (frameworks and CDRs) of large repertoires of human variable antibody domains. Our MutaGen process uses low-fidelity human polymerases, known as mutases, suggested to be involved in the somatic hypermutation process of immunoglobulin genes. Depending on the mutases used, we generated complementary mutation patterns with randomly distributed mutations. The libraries were generated with an average of 1.8 mutations per 100 amino acids. The hyperdiversified antibody fragment libraries constructed with our process should enable the selection of antibody fragments specific to virtually any target.     

Molecular and structural discrimination of proline racemase and hydroxyproline-2-epimerase from nosocomial and bacterial pathogens

The first eukaryotic proline racemase (PRAC), isolated from the human Trypanosoma cruzi pathogen, is a validated therapeutic target against Chagas' disease. This essential enzyme is implicated in parasite life cycle and infectivity and its ability to trigger host B-cell nonspecific hypergammaglobulinemia contributes to parasite evasion and persistence. Using previously identified PRAC signatures and data mining we present the identification and characterization of a novel PRAC and five hydroxyproline epimerases (HyPRE) from pathogenic bacteria. Single-mutation of key HyPRE catalytic cysteine abrogates enzymatic activity supporting the presence of two reaction centers per homodimer. Furthermore, evidences are provided that Brucella abortus PrpA [for 'proline racemase' virulence factor A] and homologous proteins from two Brucella spp are bona fide HyPREs and not 'one way' directional PRACs as described elsewhere. Although the mechanisms of aminoacid racemization and epimerization are conserved between PRAC and HyPRE, our studies demonstrate that substrate accessibility and specificity partly rely on constraints imposed by aromatic or aliphatic residues distinctively belonging to the catalytic pockets. Analysis of PRAC and HyPRE sequences along with reaction center structural data disclose additional valuable elements for in silico discrimination of the enzymes. Furthermore, similarly to PRAC, the lymphocyte mitogenicity displayed by HyPREs is discussed in the context of bacterial metabolism and pathogenesis. Considering tissue specificity and tropism of infectious pathogens, it would not be surprising if upon infection PRAC and HyPRE play important roles in the regulation of the intracellular and extracellular amino acid pool profiting the microrganism with precursors and enzymatic pathways of the host.     

Mrc1, a non-essential DNA replication protein,is required for telomere end protection following loss of capping by Cdc13, Yku or telomerase

Proteins involved in telomere end protection have previously been identified. In Saccharomyces cerevisiae, Cdc13, Yku and telomerase, mainly, prevent telomere uncapping, thus providing telomere stability and avoiding degradation and death by senescence. Here, we report that in the absence of Mrc1, a component of the replication forks, telomeres of cdc13 or yku70 mutants exhibited increased degradation, while telomerase-negative cells displayed accelerated senescence. Moreover, deletion of MRC1 increased the single-strandedness of the telomeres in cdc13-1 and yku70Δ mutant strains. An mrc1 deletion strain also exhibited slight but stable telomere shortening compared to a wild-type strain. Loss of Mrc1’s checkpoint function alone did not provoke synthetic growth defects in combination with the cdc13-1 mutation. Combinations between the cdc13-1 mutation and deletion of either TOF1 or PSY2, coding for proteins physically interacting with Mrc1, also resulted in a synthetic growth defect. Thus, the present data suggest that non-essential components of the DNA replication machinery, such as Mrc1 and Tof1, may have a role in telomere stability in addition to their role in fork progression.     

The interplay of RecA-related proteins and the MND1-HOP2 complex during meiosis in Arabidopsis thaliana

During meiosis, homologous chromosomes recognize each other, align, and exchange genetic information. This process requires the action of RecA-related proteins Rad51 and Dmc1 to catalyze DNA strand exchanges. The Mnd1-Hop2 complex has been shown to assist in Dmc1-dependent processes. Furthermore, higher eukaryotes possess additional RecA-related proteins, like XRCC3, which are involved in meiotic recombination. However, little is known about the functional interplay between these proteins during meiosis. We investigated the functional relationship between AtMND1, AtDMC1, AtRAD51, and AtXRCC3 during meiosis in Arabidopsis thaliana. We demonstrate the localization of AtMND1 to meiotic chromosomes, even in the absence of recombination, and show that AtMND1 loading depends exclusively on AHP2, the Arabidopsis Hop2 homolog. We provide evidence of genetic interaction between AtMND1, AtDMC1, AtRAD51, and AtXRCC3. In vitro assays suggest that this functional link is due to direct interaction of the AtMND1-AHP2 complex with AtRAD51 and AtDMC1. We show that AtDMC1 foci accumulate in the Atmnd1 mutant, but are reduced in number in Atrad51 and Atxrcc3 mutants. This study provides the first insights into the functional differences of AtRAD51 and AtXRCC3 during meiosis, demonstrating that AtXRCC3 is dispensable for AtDMC1 focus formation in an Atmnd1 mutant background, whereas AtRAD51 is not. These results clarify the functional interactions between key players in the strand exchange processes during meiotic recombination. Furthermore, they highlight a direct interaction between MND1 and RAD51 and show a functional divergence between RAD51 and XRCC3.     

Characterization of reemerging chikungunya virus

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.     

Phosphatidylethanol accumulation promotes intestinal hyperplasia by inducing ZONAB-mediated cell density increase in response to chronic ethanol exposure

Chronic alcohol consumption is associated with increased risk of gastrointestinal cancer. High concentrations of ethanol trigger mucosal hyperregeneration, disrupt cell adhesion, and increase the sensitivity to carcinogens. Most of these effects are thought to be mediated by acetaldehyde, a genotoxic metabolite produced from ethanol by alcohol dehydrogenases. Here, we studied the role of low ethanol concentrations, more likely to mimic those found in the intestine in vivo, and used intestinal cells lacking alcohol dehydrogenase to identify the acetaldehyde-independent biological effects of ethanol. Under these conditions, ethanol did not stimulate the proliferation of nonconfluent cells, but significantly increased maximal cell density. Incorporation of phosphatidylethanol, produced from ethanol by phospholipase D, was instrumental to this effect. Phosphatidylethanol accumulation induced claudin-1 endocytosis and disrupted the claudin-1/ZO-1 association. The resulting nuclear translocation of ZONAB was shown to mediate the cell density increase in ethanol-treated cells. In vivo, incorporation of phosphatidylethanol and nuclear translocation of ZONAB correlated with increased proliferation in the colonic epithelium of ethanol-fed mice and in adenomas of chronic alcoholics. Our results show that phosphatidylethanol accumulation after chronic ethanol exposure disrupts signals that normally restrict proliferation in highly confluent intestinal cells, thus facilitating abnormal intestinal cell proliferation.     

Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture-based biomedical applications

Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K _D of 108 ± 2 nM and has a high thermostability (T _m = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications.     

Sugars en route to the roots. Transport,metabolism and storage within plant roots and towards microorganisms of the rhizosphere

In plants, the root is a typical sink organ that relies exclusively on the import of sugar from the aerial parts. Sucrose is delivered by the phloem to the most distant root tips and, en route to the tip, is used by the different root tissues for metabolism and storage. Besides, a certain portion of this carbon is exuded in the rhizosphere, supplied to beneficial microorganisms and diverted by parasitic microbes. The transport of sugars toward these numerous sinks either occurs symplastically through cell connections (plasmodesmata) or is apoplastically mediated through membrane transporters (MST, mononsaccharide tranporters, SUT/SUC, H+/sucrose transporters and SWEET, Sugar will eventually be exported transporters) that control monosaccharide and sucrose fluxes. Here, we review recent progresses on carbon partitioning within and outside roots, discussing membrane transporters involved in plant responses to biotic and abiotic factors.