Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells

Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.     

Antimicrobial resistance and class I integrons in Salmonella enterica isolates from wild boars and Bísaro pigs

The antibiotic resistance phenotype and genotype and the integron type were characterized in 58 Salmonella enterica isolates recovered from Bísaro pigs and wild boars (20 S. Typhimurium, 17 S. Rissen, 14 S. Enteritidis and 7 S. Havana). Most S. Typhimurium isolates (15/20 of Bísaro pigs and wild boars) showed ampicillin, chloramphenicol, streptomycin, tetracycline, sulfonamide, and amoxicillin-clavulanic acid resistances. Of the 17 S. Rissen isolates of both origins, 13 were resistant to ampicillin, tetracycline and trimethoprim-sulfamethoxazole. Among the S. Enteritidis isolates of Bísaro pigs, eight were nalidixic acid-resistant and three were sulfonamide-resistant. The tet(A) or tet(G) genes were detected in most tetracycline-resistant isolates. The intI1 gene was identified in 72.5% of S. enterica isolates in which the conserved region 3' of class 1 integrons (qacEΔ1+sul1) was also amplified, whereas none had the intI2 gene. The dfrA12+orfF+aadA2 gene cassette arrangement was found in the variable region of class 1 integrons in 14 S. Rissen isolates. Fifteen S. Typhimurium isolates had two integrons with variable regions of 1000 and 1200 bp that harbored the aadA2 and blaPSE-1 gene cassettes, respectively. In these isolates the floR and tet(G) genes were also amplified, indicative of the genomic island 1 (SGI1). Salmonella Typhimurium and S. Rissen of animal origin frequently show a multi-antimicrobial resistant phenotype, which may have implications in public health.     

P2RY1/ALK3-Expressing Cells within the Adult Human Exocrine Pancreas Are BMP-7 Expandable and Exhibit Progenitor-like Characteristics

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Microbial interactions in alcoholic beverages

International Microbiology - This review examines the different types of interactions between the microorganisms involved in the fermentation processes of alcoholic beverages produced all over the...     

Changes in native and introduced host–parasite networks

Biological Invasions - Introduced species can alter the dynamics and structure of a native community. Network analysis provides a tool to study host–parasite interactions that can help to...     

The empowerment of translational research: lessons from laminopathies

The need for a collaborative approach to complex inherited diseases collectively referred to as laminopathies, encouraged Italian researchers, geneticists, physicians and patients to join in the Italian Network for Laminopathies, in 2009. Here, we highlight the advantages and added value of such a multidisciplinary effort to understand pathogenesis, clinical aspects and try to find a cure for Emery-Dreifuss muscular dystrophy, Mandibuloacral dysplasia, Hutchinson-Gilford Progeria and forms of lamin-linked cardiomyopathy, neuropathy and lipodystrophy.     

Cryopreserved CD90+ cells obtained from mobilized peripheral blood in sheep: a new source of mesenchymal stem cells for preclinical applications

Mobilized peripheral blood (MPB) bone marrow cells possess the potential to differentiate into a variety of mesenchymal tissue types and offer a source of easy access for obtaining stem cells for the development of experimental models with applications in tissue engineering. In the present work, we aimed to isolate by magnetic activated cell sorting CD90+ cells from MPB by means of the administration of Granulocyte-Colony Stimulating Factor and to evaluate cell proliferation capacity, after thawing of the in vitro culture of this population of mesenchymal stem cells (MSCs) in sheep. We obtained a median of 8.2 ± 0.6 million of CD90+ cells from the 20-mL MPB sample. After thawing, at day 15 under in vitro culture, the mean CD90+ cells determined by flow cytometry was 92.92 ± 1.29 % and cell duplication time determined by crystal violet staining was 47.59 h. This study describes for the first time the isolation, characterization, and post-in vitro culture thawing of CD90+ MSCs from mobilized peripheral blood in sheep. This population can be considered as a source of MSCs for experimental models in tissue engineering research.