Identification of an altered peptide ligand based on the endogenously presented, rheumatoid arthritis-associated, human cartilage glycoprotein-39(263-275) epitope: an MHC anchor variant peptide for immune modulation

We sought to identify an altered peptide ligand (APL) based on the endogenously expressed synovial auto-epitope of human cartilage glycoprotein-39 (HC gp-39) for modulation of cognate, HLA-DR4-restricted T cells. For this purpose we employed a panel of well-characterized T cell hybridomas generated from HC gp-39-immunized HLA-DR4 transgenic mice. The hybridomas all respond to the HC gp-39(263-275) epitope when bound to HLA-DR4(B1*0401) but differ in their fine specificities. First, the major histocompatibility complex (MHC) and T-cell receptor (TCR) contact residues were identified by analysis of single site substituted analogue peptides for HLA-DR4 binding and cognate T cell recognition using both T hybridomas and polyclonal T cells from peptide-immunized HLA-DR4 transgenic mice. Analysis of single site substituted APL by cognate T cells led to identification of Phe265 as the dominant MHC anchor. The amino acids Ala268, Ser269, Glu271 and Thr272 constituted the major TCR contact residues, as substitution at these positions did not affect HLA-DR4(B1*0401) binding but abrogated T cell responses. A structural model for visualisation of TCR recognition was derived. Second, a set of non-classical APLs, modified at the MHC key anchor position but with unaltered TCR contacts, was developed. When these APLs were analysed, a partial TCR agonist was identified and found to modulate the HC gp-39(263-275)-specific, pro-inflammatory response in HLA-DR4 transgenic mice. We identified a non-classical APL by modification of the p1 MHC anchor in a synovial auto-epitope. This APL may qualify for rheumatoid arthritis immunotherapy.     

129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant

Mice derived from the 129 strain have a nonsense codon mutation in exon 2 of the polymerase iota (Polι) gene and are therefore considered Polι deficient. When we amplified Polι mRNA from 129/SvJ or 129/Ola testes, only a small fraction of the full-length cDNA contained the nonsense mutation; the major fraction corresponded to a variant Polι isoform lacking exon 2. Polι mRNA lacking exon 2 contains an open reading frame, and the corresponding protein was detected using a polyclonal antibody raised against the C terminus of the murine Polι protein. The identity of the corresponding protein was further confirmed by mass spectrometry. Although the variant protein was expressed at only 5 to 10% of the level of wild-type Polι, it retained de novo DNA synthesis activity, the capacity to form replication foci following UV irradiation, and the ability to rescue UV light sensitivity in Polι^−/− embryonic fibroblasts derived from a new, fully deficient Polι knockout (KO) mouse line. Furthermore, in vivo treatment of 129-derived male mice with Velcade, a drug that inhibits proteasome function, stabilized and restored a substantial amount of the variant Polι in these animals, indicating that its turnover is controlled by the proteasome. An analysis of two xeroderma pigmentosum-variant (XPV) cases corresponding to missense mutants of Polη, a related translesion synthesis (TLS) polymerase in the same family, similarly showed a destabilization of the catalytically active mutant protein by the proteasome. Collectively, these data challenge the prevailing hypothesis that 129-derived strains of mice are completely deficient in Polι activity. The data also document, both for 129-derived mouse strains and for some XPV patients, new cases of genetic defects corresponding to the destabilization of an otherwise functional protein, the phenotype of which is reversible by proteasome inhibition.     

Ecological restoration of rich fens in Europe and North America: from trial and error to an evidence‐based approach

Fens represent a large array of ecosystem services, including the highest biodiversity found among wetlands, hydrological services, water purification and carbon sequestration. Land‐use change and drainage has severely damaged or annihilated these services in many parts of North America and Europe; restoration plans are urgently needed at the landscape level. We review the major constraints on the restoration of rich fens and fen water bodies in agricultural areas in Europe and disturbed landscapes in North America: (i) habitat quality problems: drought, eutrophication, acidification, and toxicity, and (ii) recolonization problems: species pools, ecosystem fragmentation and connectivity, genetic variability, and invasive species; and here provide possible solutions. We discuss both positive and negative consequences of restoration measures, and their causes. The restoration of wetland ecosystem functioning and services has, for a long time, been based on a trial‐and‐error approach. By presenting research and practice on the restoration of rich fen ecosystems within agricultural areas, we demonstrate the importance of biogeochemical and ecological knowledge at different spatial scales for the management and restoration of biodiversity, water quality, carbon sequestration and other ecosystem services, especially in a changing climate. We define target processes that enable scientists, nature managers, water managers and policy makers to choose between different measures and to predict restoration prospects for different types of deteriorated fens and their starting conditions.     

Modelling sugar diffusion across plant leaf cuticles: the effect of free water on substrate availability to phyllosphere bacteria

We present a continuous model for the diffusion of sugars across intact plant leaf cuticles. It is based on the flow of sugars from a source, representing the leaf apoplast, to a sink, in the shape of a hemispherical drop of water on the outside of the cuticle. Flow is a function of the difference between sugar concentrations C_Source and C_Sink, permeability P of the cuticle, volume V_Sink of the water drop, as well as its contact angle α with the cuticle surface. Using a bacterial bioreporter for fructose, and a two‐compartment experimental set‐up consisting of isolated cuticles of walnut (Juglans regia) carrying water droplets while floating on solutions with increasing concentrations of fructose, we determined a value of 1 × 10^?6 m h^?1 for P. Using this value, we explored different scenarios for the leaching of sugars across plant leaf cuticles to reveal in quantitative terms how diffusion takes longer when V_Sink increases, P decreases or α increases. Bacterial growth was modelled as a function of changes in P, α and V_Sink and was consistent with observations or suggestions from the literature in relation to the availability of free water on leaves. These results are discussed in the light of bacteria as ecosystem engineers, i.e. with the ability to modify the plant leaf surface environment in favour of their own survival, e.g. by increasing cuticle leakage or leaf wetness. Our model represents a first step towards a more comprehensive model which will enhance our quantitative understanding of the factors that play a role in nutrient availability to bacterial colonizers of the phyllosphere, or plant leaf surface.     

Gain of function of mutant p53 by coaggregation with multiple tumor suppressors

Many p53 missense mutations possess dominant-negative activity and oncogenic gain of function. We report that for structurally destabilized p53 mutants, these effects result from mutant-induced coaggregation of wild-type p53 and its paralogs p63 and p73, thereby also inducing a heat-shock response. Aggregation of mutant p53 resulted from self-assembly of a conserved aggregation-nucleating sequence within the hydrophobic core of the DNA-binding domain, which becomes exposed after mutation. Suppressing the aggregation propensity of this sequence by mutagenesis abrogated gain of function and restored activity of wild-type p53 and its paralogs. In the p53 germline mutation database, tumors carrying aggregation-prone p53 mutations have a significantly lower frequency of wild-type allele loss as compared to tumors harboring nonaggregating mutations, suggesting a difference in clonal selection of aggregating mutants. Overall, our study reveals a novel disease mechanism for mutant p53 gain of function and suggests that, at least in some respects, cancer could be considered an aggregation-associated disease.     

Linoleate isomerase activity occurs in lactic acid bacteria strains and is affected by pH and temperature

Aims: To investigate the ability of lactic acid bacteria (LAB) to convert linoleic acid (LA) and α‐linolenic acid (α‐LNA) to conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), respectively. To assess pH and temperature influences on CLA and CLNA production by Lactobacillus sakei LMG 13558. Methods and Results: A screening of 48 LAB yielded one Lactobacillus curvatus, five Lactobacillus plantarum and four Lact. sakei strains displaying linoleate isomerase (LAI) activity. CLNA conversion percentages varied largely (1–60%). CLA conversion, occurring in three strains, was lower (2–5%). The LAI gene sequences of the ten LAI‐positive strains shared 75–99% identity with the LAI gene sequence of a Lact. plantarum AS1.555. At pH 6·2, CLA and CLNA production by Lact. sakei LMG 13558 was higher at 30°C than at 20 and 25°C. At pH 5·5 (30°C) or 37°C (pH 6·2), LA was not converted and α‐LNA only slightly converted. Conclusions: LAB show strain‐dependent LAI activity. Production of CLA and CLNA is affected by pH and temperature, as shown for Lact. sakei LMG 13558. Significance and Impact of the Study: Several LAB produce CLA and/or CLNA, as shown for Lact. sakei and Lact. curvatus for the first time. These findings offer potential for the manufacturing of fermented functional foods.     

In situ ESBL conjugation from avian to human Escherichia coli during cefotaxime administration

Aims: The behaviour of an Escherichia coli isolate of broiler origin harbouring a bla_TEM‐52‐carrying plasmid (lactose‐negative mutant of B1‐54, IncII group) was studied in an in situ continuous flow culture system, simulating the human caecum and the ascending colon during cefotaxime administration. Methods and Results: Fresh faeces from a healthy volunteer, negative for cephalosporin‐resistant E. coli, were selected to prepare inocula. The microbiota was monitored by plating on diverse selective media, and a shift in the populations of bacteria was examined by 16S rDNA PCR denaturing gradient gel electrophoresis. Escherichia coli transconjugants were verified by plasmid and pulsed‐field gel electrophoresis profiles (PFGE). The avian extended‐spectrum β‐lactamase‐positive E. coli was able to proliferate without selective pressure of cefotaxime, and E. coli transconjugants of human origin were detected 24 h after inoculation of the donor strain. Upon administration of cefotaxime to the fresh medium, an increase in the population size of E. coli B1‐54 and the transconjugants was observed. PFGE and plasmid analysis revealed a limited number of human E. coli clones receptive for the bla_TEM‐52‐carrying plasmid. Conclusions: These observations provide evidence of the maintenance of an E. coli strain of poultry origin and the horizontal gene transfer in the human commensal bowel microbiota even without antimicrobial treatment. Significance and Impact of the Study: The fact that an E. coli strain of poultry origin might establish itself and transfer its bla gene to commensal human E. coli raises public health concerns.     

An ensemble biclustering approach for querying gene expression compendia with experimental lists

MOTIVATION: Query-based biclustering techniques allow interrogating a gene expression compendium with a given gene or gene list. They do so by searching for genes in the compendium that have a profile close to the average expression profile of the genes in this query-list. As it can often not be guaranteed that the genes in a long query-list will all be mutually coexpressed, it is advisable to use each gene separately as a query. This approach, however, leaves the user with a tedious post-processing of partially redundant biclustering results. The fact that for each query-gene multiple parameter settings need to be tested in order to detect the 'most optimal bicluster size' adds to the redundancy problem. RESULTS: To aid with this post-processing, we developed an ensemble approach to be used in combination with query-based biclustering. The method relies on a specifically designed consensus matrix in which the biclustering outcomes for multiple query-genes and for different possible parameter settings are merged in a statistically robust way. Clustering of this matrix results in distinct, non-redundant consensus biclusters that maximally reflect the information contained within the original query-based biclustering results. The usefulness of the developed approach is illustrated on a biological case study in Escherichia coli. Availability and implementation: Compiled Matlab code is available from http://homes.esat.kuleuven.be/~kmarchal/Supplementary_Information_DeSmet_2011/.