On a general model structure for macroscopic biological reaction rates

Macroscopic modelling of bioprocesses requires the determination of a biological reaction scheme and a kinetic model. The a priori selection of an appropriate kinetic model structure is usually made difficult by the lack of detailed bioprocess knowledge and the profusion of apparently similar biological kinetic laws. Moreover, parameter identification is made arduous and time-consuming by the strong non-linearities involved in kinetic laws. In most cases, these kinetic structures are non-linearizable and no first parameter estimation can be deduced easily. In order to avoid such identification problems, Bogaerts et al. [Bogaerts, Ph., Castillo, J., Hanus, R., 1999. A general mathematical modelling technique for bioprocesses in engineering applications. Syst. Anal. Model. Simul. 35, 87-113] have developed a general linearizable kinetic structure which allows the representation of activation and/or inhibition effects of each component in the culture. This paper further generalizes this structure in order to improve the way saturation effects are taken into account, and in turn, improve the biological interpretation of the model parameters. The main advantage of the proposed structure lies in an associated systematic estimation procedure. The usefulness of the proposed model is tested with simulated as well as with experimental data.     

Principles and practice in ethical review of animal experiments across Europe: summary of the report of a FELASA working group on ethical evaluation of animal experiments

This paper summarizes a more detailed report produced by the Federation of European Laboratory Animal Science Associations (FELASA 2005), which describes and explores a set of principles for the conduct of ethical review of laboratory animal use. It presents a synopsis of results from a questionnaire that elicited information on how each of 20 countries represented in FELASA currently approaches such ethical review. This information suggests that, although local practices differ, there is an emerging consensus on the key elements that any ethical review process should involve. Drawing on the questionnaire findings, this summary also includes a brief discussion to support and amplify a series of recommendations, covering the objectives of ethical review; legal requirements; the scope of work reviewed and the 'level' at which review is approached; general principles for the organization of ethical review processes; the factors considered in the review; needs for ongoing review after initial authorization; participants in the review process; wider impacts of the review process; and strategies that can help to ensure quality and consistency of review outcomes. For further information and examples of current practice, as well as more detailed discussion to support the recommendations, readers are urged to refer to the complete report, available at http://www.lal.org.uk/pdffiles/FELASA_ethics_FULL_Report. pdf or via: http://www.felasa.eu/recommendations.htm.     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Somatic CTG•CAG repeat instability in a mouse model for myotonic dystrophy type 1 is associated with changes in cell nuclearity and DNA ploidy

Background  

Trinucleotide instability is a hallmark of degenerative neurological diseases like Huntington's disease, some forms of spinocerebellar ataxia and myotonic dystrophy type 1 (DM1). To investigate the effect of cell type and cell state on the behavior of the DM1 CTG•CAG repeat, we studied a knock-in mouse model for DM1 at different time points during ageing and followed how repeat fate in cells from liver and pancreas is associated with polyploidization and changes in nuclearity after the onset of terminal differentiation.     

The de-ubiquitylating enzymes USP26 and USP37 regulate homologous recombination by counteracting RAP80

The faithful repair of DNA double-strand breaks (DSBs) is essential to safeguard genome stability. DSBs elicit a signaling cascade involving the E3 ubiquitin ligases RNF8/RNF168 and the ubiquitin-dependent assembly of the BRCA1-Abraxas-RAP80-MERIT40 complex. The association of BRCA1 with ubiquitin conjugates through RAP80 is known to be inhibitory to DSB repair by homologous recombination (HR). However, the precise regulation of this mechanism remains poorly understood. Through genetic screens we identified USP26 and USP37 as key de-ubiquitylating enzymes (DUBs) that limit the repressive impact of RNF8/RNF168 on HR. Both DUBs are recruited to DSBs where they actively remove RNF168-induced ubiquitin conjugates. Depletion of USP26 or USP37 disrupts the execution of HR and this effect is alleviated by the simultaneous depletion of RAP80. We demonstrate that USP26 and USP37 prevent excessive spreading of RAP80-BRCA1 from DSBs. On the other hand, we also found that USP26 and USP37 promote the efficient association of BRCA1 with PALB2. This suggests that these DUBs limit the ubiquitin-dependent sequestration of BRCA1 via the BRCA1-Abraxas-RAP80-MERIT40 complex, while promoting complex formation and cooperation of BRCA1 with PALB2-BRCA2-RAD51 during HR. These findings reveal a novel ubiquitin-dependent mechanism that regulates distinct BRCA1-containing complexes for efficient repair of DSBs by HR.     

Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains

Lager brewing strains of Saccharomyces pastorianus are natural interspecific hybrids originating from the spontaneous hybridization of Saccharomyces cerevisiae and Saccharomyces eubayanus. Over the past 500 years, S. pastorianus has been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics of S. pastorianus genomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed that S. pastorianus strain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants in S. pastorianus CBS1483. Analysis of eight additional S. pastorianus strains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes.     

Strategies for bacterial expression of protein-peptide complexes: application to solubilization of papillomavirus E6

E6 is a small oncoprotein involved in tumorigenesis induced by papillomaviruses (PVs). E6 often recognizes its cellular targets by binding to short motifs presenting the consensus LXXLL. E6 proteins have long resisted structural analysis. We found that bovine papillomavirus type 1 (BPV1) E6 binds the N-terminal LXXLL motif of the cellular protein paxillin with significantly higher affinity as compared to other E6/peptide interactions. Although recombinant BPV1 E6 was poorly soluble in the free state, provision of the paxillin LXXLL peptide during BPV1 E6 biosynthesis greatly enhanced the protein's solubility. Expression of BPV1 E6/LXXLL peptide complexes was carried out in bacteria in the form of triple fusion constructs comprising, from N- to C-terminus, the soluble carrier protein maltose binding protein (MBP), the LXXLL motif and the E6 protein. A TEV protease cleavage site was placed either between MBP and LXXLL motif or between LXXLL motif and E6. These constructs allowed us to produce highly concentrated samples of BPV1 E6, either covalently fused to the C-terminus of the LXXLL motif (intra-molecular complex) or non-covalently bound to it (inter-molecular complex). Heteronuclear NMR measurements were performed and showed that the E6 protein was folded with similar conformations in both covalent and non-covalent complexes. These data open the way to novel structural and functional studies of the BPV1 E6 in complex with its preferential target motif.     

MAGE-C2/CT10 protein expression is an independent predictor of recurrence in prostate cancer

The cancer-testis (CT) family of antigens is expressed in a variety of malignant neoplasms. In most cases, no CT antigen is found in normal tissues, except in testis, making them ideal targets for cancer immunotherapy. A comprehensive analysis of CT antigen expression has not yet been reported in prostate cancer. MAGE-C2/CT-10 is a novel CT antigen. The objective of this study was to analyze extent and prognostic significance of MAGE-C2/CT10 protein expression in prostate cancer. 348 prostate carcinomas from consecutive radical prostatectomies, 29 castration-refractory prostate cancer, 46 metastases, and 45 benign hyperplasias were immunohistochemically analyzed for MAGE-C2/CT10 expression using tissue microarrays. Nuclear MAGE-C2/CT10 expression was identified in only 3.3% primary prostate carcinomas. MAGE-C2/CT10 protein expression was significantly more frequent in metastatic (16.3% positivity) and castration-resistant prostate cancer (17% positivity; p<0.001). Nuclear MAGE-C2/CT10 expression was identified as predictor of biochemical recurrence after radical prostatectomy (p = 0.015), which was independent of preoperative PSA, Gleason score, tumor stage, and surgical margin status in multivariate analysis (p<0.05). MAGE-C2/CT10 expression in prostate cancer correlates with the degree of malignancy and indicates a higher risk for biochemical recurrence after radical prostatectomy. Further, the results suggest MAGE-C2/CT10 as a potential target for adjuvant and palliative immunotherapy in patients with prostate cancer.