From DNA sequence to application: possibilities and complications

The development of sophisticated genetic tools during the past 15 years have facilitated a tremendous increase of fundamental and application-oriented knowledge of lactic acid bacteria (LAB) and their bacteriophages. This knowledge relates both to the assignments of open reading frames (ORF’s) and the function of non-coding DNA sequences. Comparison of the complete nucleotide sequences of several LAB bacteriophages has revealed that their chromosomes have a fixed, modular structure, each module having a set of genes involved in a specific phase of the bacteriophage life cycle. LAB bacteriophage genes and DNA sequences have been used for the construction of temperature-inducible gene expression systems, gene-integration systems, and bacteriophage defence systems. The function of several LAB open reading frames and transcriptional units have been identified and characterized in detail. Many of these could find practical applications, such as induced lysis of LAB to enhance cheese ripening and re-routing of carbon fluxes for the production of a specific amino acid enantiomer. More knowledge has also become available concerning the function and structure of non-coding DNA positioned at or in the vicinity of promoters. In several cases the mRNA produced from this DNA contains a transcriptional terminator-antiterminator pair, in which the antiterminator can be stabilized either by uncharged tRNA or by interaction with a regulatory protein, thus preventing formation of the terminator so that mRNA elongation can proceed. Evidence has accumulated showing that also in LAB carbon catabolite repression in LAB is mediated by specific DNA elements in the vicinity of promoters governing the transcription of catabolic operons. Although some biological barriers have yet to be solved, the vast body of scientific information presently available allows the construction of tailor-made genetically modified LAB. Today, it appears that societal constraints rather than biological hurdles impede the use of genetically modified LAB.

A proposal for a coordinated effort for the determination of brainwide neuroanatomical connectivity in model organisms at a mesoscopic scale

In this era of complete genomes, our knowledge of neuroanatomical circuitry remains surprisingly sparse. Such knowledge is however critical both for basic and clinical research into brain function. Here we advocate for a concerted effort to fill this gap, through systematic, experimental mapping of neural circuits at a mesoscopic scale of resolution suitable for comprehensive, brain-wide coverage, using injections of tracers or viral vectors. We detail the scientific and medical rationale and briefly review existing knowledge and experimental techniques. We define a set of desiderata, including brain-wide coverage; validated and extensible experimental techniques suitable for standardization and automation; centralized, open access data repository; compatibility with existing resources, and tractability with current informatics technology. We discuss a hypothetical but tractable plan for mouse, additional efforts for the macaque, and technique development for human. We estimate that the mouse connectivity project could be completed within five years with a comparatively modest budget.Comment: 41 page

Inverse correlation between PDGFC expression and lymphocyte infiltration in human papillary thyroid carcinomas

<p>Abstract</p> <p>Background</p> <p>Members of the PDGF family have been suggested as potential biomarkers for papillary thyroid carcinomas (PTC). However, it is known that both expression and stimulatory effect of PDGF ligands can be affected by inflammatory cytokines. We have performed a microarray study in a collection of PTCs, of which about half the biopsies contained tumour-infiltrating lymphocytes or thyroiditis. To investigate the expression level of PDGF ligands and receptors in PTC we measured the relative mRNA expression of all members of the PDGF family by qRT-PCR in 10 classical PTC, eight clinically aggressive PTC, and five non-neoplastic thyroid specimens, and integrated qRT-PCR data with microarray data to enable us to link PDGF-associated gene expression profiles into networks based on recognized interactions. Finally, we investigated potential influence on PDGF mRNA levels by the presence of tumour-infiltrating lymphocytes.</p> <p>Methods</p> <p>qRT-PCR was performed on <it>PDGFA</it>, <it>PDGFB</it>, <it>PDGFC</it>, <it>PDGFD</it>, <it>PDGFRA PDGFRB </it>and a selection of lymphocyte specific mRNA transcripts. Semiquantitative assessment of tumour-infiltrating lymphocytes was performed on the adjacent part of the biopsy used for RNA extraction for all biopsies, while direct quantitation by qRT-PCR of lymphocyte-specific mRNA transcripts were performed on RNA also subjected to expression analysis. Relative expression values of PDGF family members were combined with a cDNA microarray dataset and analyzed based on clinical findings and PDGF expression patterns. Ingenuity Pathway Analysis (IPA) was used to elucidate potential molecular interactions and networks.</p> <p>Results</p> <p>PDGF family members were differentially regulated at the mRNA level in PTC as compared to normal thyroid specimens. Expression of <it>PDGFA </it>(p = 0.003), <it>PDGFB </it>(p = 0.01) and <it>PDGFC </it>(p = 0.006) were significantly up-regulated in PTCs compared to non-neoplastic thyroid tissue. In addition, expression of <it>PDGFC </it>was significantly up-regulated in classical PTCs as compared to clinically aggressive PTCs (p = 0.006), and <it>PDGFRB </it>were significantly up-regulated in clinically aggressive PTCs (p = 0.01) as compared to non-neoplastic tissue. Semiquantitative assessment of lymphocytes correlated well with quantitation of lymphocyte-specific gene expression. Further more, by combining TaqMan and microarray data we found a strong inverse correlation between <it>PDGFC </it>expression and the expression of lymphocyte specific mRNAs.</p> <p>Conclusion</p> <p>At the mRNA level, several members of the PDGF family are differentially expressed in PTCs as compared to normal thyroid tissue. Of these, only the <it>PDGFC </it>mRNA expression level initially seemed to distinguish classical PTCs from the more aggressive PTCs. However, further investigation showed that <it>PDGFC </it>expression level correlated inversely to the expression of several lymphocyte specific genes, and to the presence of lymphocytes in the biopsies. Thus, we find that <it>PDGFC </it>mRNA expression were down-regulated in biopsies containing infiltrated lymphocytes or thyroiditis. No other PDGF family member could be linked to lymphocyte specific gene expression in our collection of PTCs biopsies.</p

Poly-β-hydroxybutyrate administration during early life: effects on performance, immunity and microbial community of European sea bass yolk-sac larvae

The reliable production of marine fish larvae is one of the major bottlenecks in aquaculture due to high mortalities mainly caused by infectious diseases. To evaluate if the compound poly-β-hydroxybutyrate (PHB) might be a suitable immunoprophylactic measure in fish larviculture, its capacity to improve immunity and performance in European sea bass (Dicentrarchus labrax) yolk-sac larvae was explored. PHB was applied from mouth opening onwards to stimulate the developing larval immune system at the earliest possible point in time. Larval survival, growth, microbiota composition, gene expression profiles and disease resistance were assessed. PHB administration improved larval survival and, furthermore, altered the larva-associated microbiota composition. The bacterial challenge test using pathogenic Vibrio anguillarum revealed that the larval disease resistance was not influenced by PHB. The expression profiles of 26 genes involved e.g. in the immune response showed that PHB affected the expression of the antimicrobial peptides ferritin (fer) and dicentracin (dic), however, the response to PHB was inconsistent and weaker than previously demonstrated for sea bass post-larvae. Hence, the present study highlights the need for more research focusing on the immunostimulation of different early developmental stages for gaining a more comprehensive picture and advancing a sustainable production of high quality fry

Characterization of genetic elements required for site-specific integration of the temperate lactococcal bacteriophage phi LC3 and construction of integration-negative phi LC3 mutants.

The genetic elements required for the integration of the temperate lactococcal bacteriophage phi LC3 into the chromosome of its bacterial host, Lactococcus lactis subsp. cremoris, were identified and characterized. The phi LC3 phage attachment site, attP, was mapped and sequenced. DNA sequence analysis of attP and of the bacterial attachment site, attB, as well as the two phage-host junctions, attR and attL, in the chromosome of a phi LC3 lysogen, identified a 9-bp common core region, 5'-TTCTTCATG'-3, within which the strand exchange reaction takes place during integration. The attB core sequence is located within the C-terminal part of an open reading frame of unknown function. The phi LC3 integrase gene (int), encoding the phi LC3 site-specific recombinase, was identified and is located adjacent to attP. The phi LC3 Int protein, as deduced from the nucleotide sequence, is a basic protein of 374 amino acids that shares significant sequence similarity with other site-specific recombinases of the integrase family. Phage phi LC3 int- and int-attP-defective mutants, conferring an abortive lysogenic phenotype, were constructed

A novel library-independent approach based on high-throughput cultivation in Bioscreen and fingerprinting by FTIR spectroscopy for microbial source tracking in food industry

Microbiological source tracking (MST) for food industry is a rapid growing area of research and technology development. In this paper, a new library-independent approach for MST is presented. It is based on a high-throughput liquid microcultivation and FTIR spectroscopy. In this approach, FTIR spectra obtained from micro-organisms isolated along the production line and a product are compared to each other. We tested and evaluated the new source tracking approach by simulating a source tracking situation. In this simulation study, a selection of 20 spoilage mould strains from a total of six genera (Alternaria, Aspergillus, Mucor, Paecilomyces, Peyronellaea and Phoma) was used. The simulation of the source tracking situation showed that 80–100% of the sources could be correctly identified with respect to genus/species level. When performing source tracking simulations, the FTIR identification diverged for Phoma glomerata strain in the reference collection. When reidentifying the strain by sequencing, it turned out that the strain was a Peyronellaea arachidicola. The obtained results demonstrated that the proposed approach is a versatile tool for identifying sources of microbial contamination. Thus, it has a high potential for routine control in the food industry due to low costs and analysis time. Significance and Impact of the Study The source tracking of fungal contamination in the food industry is an important aspect of food safety. Currently, all available methods are time consuming and require the use of a reference library that may limit the accuracy of the identification. In this study, we report for the first time, a library-independent FTIR spectroscopic approach for MST of fungal contamination along the food production line. It combines high-throughput microcultivation and FTIR spectroscopy and is specific on the genus and species level. Therefore, such an approach possesses great importance for food safety control in food industry.publishedVersio