Stepwise prediction of conformational discontinuous B-cell epitopes using the Mapitope algorithm

Mapping the epitope of an antibody is of great interest, since it contributes much to our understanding of the mechanisms of molecular recognition and provides the basis for rational vaccine design. Here we present Mapitope, a computer algorithm for epitope mapping. The algorithm input is a set of affinity isolated peptides obtained by screening phage display peptide-libraries with the antibody of interest. The output is usually 1-3 epitope candidates on the surface of the atomic structure of the antigen. We have systematically tested the performance of Mapitope by assessing the effect of the algorithm parameters on the final prediction. Thus, we have examined the effect of the statistical threshold (ST) parameter, relating to the frequency distribution and enrichment of amino acid pairs from the isolated peptides and the D (distance) and E (exposure) parameters which relate to the physical parameters of the antigen. Two model systems were analyzed in which the antibody of interest had previously been co-crystallized with the antigen and thus the epitope is a given. The Mapitope algorithm successfully predicted the epitopes in both models. Accordingly, we formulated a stepwise paradigm for the prediction of discontinuous conformational epitopes using peptides obtained from screening phage display libraries. We applied this paradigm to successfully predict the epitope of the Trastuzumab antibody on the surface of the Her-2/neu receptor in a third model system.     

A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.     

Specific and Selective Bacteriophages in the Fight against Multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii causes serious infections especially in immunocompromised and/or hospitalized patients. Several A. baumannii strains are multidrug resistant and infect wounds, bones, and the respiratory tract. Current studies are focused on finding new effective agents against A. baumannii. Phage therapy is a promising means to fight this bacterium and many studies on procuring and applying new phages against A. baumannii are currently being conducted. As shown in animal models, phages against multidrug-resistant A. baumannii may control bacterial infections caused by this pathogen and may be a real hope to solve this dangerous health problem.     

Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.     

Continual assembly of desmosomes within stable intercellular contacts of epithelial A-431 cells

Subclones of human carcinoma-derived A-431 cell line stably producing fusion proteins consisting of the enhanced green fluorescent protein and either human desmoglein 2 (Dsg-GFP) or human plakoglobin (GFP-Pg) were used to examine the behavior of desmosomes in living cells. Immunofluorescence microscopy of the fixed cells showed that both fusion proteins, which were expressed in significantly lower levels relative to their endogenous counterparts, were efficiently recruited into desmosomes. Time-lapse confocal imaging of these cells reveals that such GFP-labeled desmosomes (GFP desmosomes) are stable structures which exhibit various dynamic and motile activities. The most notable are independent lateral mobility and fusion. Furthermore, the continual assembly of new nascent desmosomes is observed within stable contacts located at the middle of the epithelial sheet. A new GFP desmosome appears as a closely apposed group of fine patches which after a few minutes aggregate into a single structure. These three dynamic processes resulted in constant changes of desmosome distribution, numbers, and sizes. In addition, fluorescence recovery after photobleaching experiments showed that fine patches of desmosomal proteins may participate in desmosome maintenance. Such a diverse range of dynamic activities of desmosomes apparently produces flexible but tight cell-cell adhesion required for different morphogenetic events in epithelial structures.This work was supported by grant AR44016-04 from the National Institutes of Health     

Ultrasensitive nucleic acid biosensors for early cancer diagnostics

We have developed ultrasensitive nucleic acid detection systems involving an amplification step where the analytical signal correlates directly to the amount of nucleic acid in the solution So far, we have performed nucleic acid quantification on several breast cancer susceptibility genes and were able to detect nucleic acid amounts that ranged from 0.1–1.0 fg of nucleic acid, which is at least 1000 times more sensitive than conventional fluorescent detection methods. The biosensors are so sensitive that they can be used for direct detection of breast cancer susceptibility genes in mRNA without involving a PCR step.     

Trainmasters or Easyjet pilots? Historical production,labor organizing,and the Spanish engine drivers’ union

This article presents an analysis of the relationship between historical memory and the emergence and reproduction of a specific model of unionism. I argue that in order to understand the militant particularism of the Spanish engine drivers, we need to look at the historical representations that it is embedded in. Recourse to a generational framework makes possible the sustained presentation of the union as a case of successful workers’ organizing, in spite of evidence to the contrary. The historical ethnographic analysis of SEMAF, the Spanish engine drivers’ union, contributes to the analysis of historical memory within the contemporary anthropology of class. The article contributes to theoretical debates in the anthropology of class by reclaiming Michel Trouillot’s conceptualization of the historical process. Two aspects of Trouillot’s work are singled out: his dynamic understanding of the process of historical production and the corollary formulation of the overlapping capacities in which people participate in it (as agents, actors, and subjects) and his emphasis on the importance of expanding scholarly views of the field of historical production.     

Critical threshold meal size and molt initiation in Rhodnius prolixus

The molting process and body growth in Rhodnius prolixus (Hemiptera: Reduviidae) (Ståhl, 1859) are significantly influenced by the availability and quality of food. Based on the body weight of each stage, the present study provides estimates of a potential critical weight threshold required for molt initiation in R. prolixus. In addition, a new measure given by the area under the weight curve is proposed, which encapsulates both body weight and time. It is shown that this measure is consistent with the data, and allows the estimation of a pre‐refractory period (i.e. the time interval between the moment at which the critical weight threshold is reached and the moment when no further meals are accepted). The present analysis estimates the critical weight threshold as 1.6, 5.3, 12.9, 42.0 and 97.0 mg for stages 1–5, respectively, whereas the values of the area under the curve threshold as 5, 16, 31.2, 159.7 and 329.9 mg days for stages 1–5, respectively. The results of the present study confirm the existence of a weight‐dependent mechanism for the initiation of molting in R. prolixus.