Review ‐ Helicobacter pylori and non‐malignant upper gastro‐intestinal diseases

This review takes into account recent publications focusing on the relationship between Helicobacter pylori infection and non‐malignant diseases of the upper gastro‐intestinal tract. The authors have summarized current knowledge on associations between the H pylori infection and non‐malignant upper GI conditions including gastroesophageal reflux disease (GERD), Barrett's esophagus, eosinophilic esophagitis (EOE), peptic ulcer disease (PUD), H pylori gastritis, celiac disease and functional dyspepsia. In the field of GERD, current data focusing on different locations of H pylori infection detect significant differences between antrum‐ and corpus predominant gastritis explainable by different changes in acid secretion in different gastric niches. High volume studies from Sweden and Brazil underline the safety of H pylori eradication concerning the risk of Barret's esophagus or adenocarcinoma. The relationship betweenH pylori infection and EOE remains uncertain, but current data supports the concept of expected positive and protective effects of H pylori exposure reducing the risk of EOE. Analyzing biomarkers might be helpful to identify subjects under risk for the development of precancerous lesions after H pylori infection, where microRNAs, IL‐9 and IL‐4, and also Tc17/9 and Th17/9 and microbiota profiles showed promising results to identify subgroups under risk.     

Generation of menangle virus nucleocapsid-like particles in yeast Saccharomyces cerevisiae

Menangle virus (MenV), which was isolated in Australia in 1997 during an outbreak of severe reproductive disease in pigs, is a novel member of the genus Rubulavirus in the family Paramyxoviridae. Although successfully eradicated from the affected piggery, fruit bats are considered to be the natural reservoir of the virus and therefore an ongoing risk of re-introduction to the pig population exists. Accordingly, reagents to facilitate serological surveillance are required to enhance the diagnostic capability for MenV, which is a newly recognized cause of disease in pigs with the potential to severely affect production in naive breeding herds. To address this need, recombinant MenV nucleocapsid (N) protein was expressed in the yeast Saccharomyces cerevisiae. Using the expression vector pFGG3 under control of the GAL7 promoter, high yields of recombinant MenV N protein were obtained. Electron microscopy demonstrated that purified recombinant N protein self-assembled into nucleocapsid-like particles which were identical in density and morphology, although not in length, to authentic nucleocapsids from virus-infected cells. Electron microscopy analysis also showed that yeast-expressed N protein which lacked the C-terminal tail (amino acid residues 400–519) formed significantly longer and denser nucleocapsid-like particles. Nucleocapsid-like particles derived from the full-length recombinant protein were stable and readily purified by CsCl gradient ultracentrifugation. When used as coating antigen in an indirect ELISA, the recombinant N protein reacted with sera derived from pigs experimentally infected with MenV and a simple serological assay to detect MenV-specific antibodies in pigs, fruit bats and humans could be designed on this basis.     

Identification of the genus Geobacillus using genus-specific primers, based on the 16S-23S rRNA gene internal transcribed spacer

The aim of this study was to develop an easy and accurate technique for the identification of the genus Geobacillus. For this purpose, Geobacillus genus-specific primers GEOBAC (GEOBAC-F and GEOBAC-R) based on the 16S-23S rRNA gene internal transcribed spacer (ITS) region sequences have been designed. In total, 52 sequences from three species of the genus Geobacillus (Geobacillus stearothermophilus, Geobacillus kaustophilus and Geobacillus lituanicus) were examined for the design of these primers. Analysis of the sequences revealed three highly conservative regions common to these species: 5' and 3' end regions of 16S-23S rRNA gene ITSs and box A. Some sequences possessed two additional conservative regions - genes of tRNA(Ile) and tRNA(Ala). These particular sequences were chosen for the construction of the primers. The designed primers targeted the gene of tRNA(Ile) and the 3' end region of ITSs. This technique was validated with both the reference strains of the genus Geobacillus and the thermophilic aerobic endospore-forming environmental isolates. Different Geobacillus species could be grouped according to the number and size of GEOBAC-PCR products and identified on the basis of the AluI and TaqI restriction analysis of these products.     

Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks

Background

Here we describe a novel cultivation method, called EnBase?, or enzyme-based-substrate-delivery, for the growth of microorganisms in millilitre and sub-millilitre scale which yields 5 to 20 times higher cell densities compared to standard methods. The novel method can be directly applied in microwell plates and shake flasks without any requirements for additional sensors or liquid supply systems. EnBase is therefore readily applicable for many high throughput applications, such as DNA production for genome sequencing, optimisation of protein expression, production of proteins for structural genomics, bioprocess development, and screening of enzyme and metagenomic libraries.

Results

High cell densities with EnBase are obtained by applying the concept of glucose-limited fed-batch cultivation which is commonly used in industrial processes. The major difference of the novel method is that no external glucose feed is required, but glucose is released into the growth medium by enzymatic degradation of starch. To cope with the high levels of starch necessary for high cell density cultivation, starch is supplied to the growing culture suspension by continuous diffusion from a storage gel. Our results show that the controlled enzyme-based supply of glucose allows a glucose-limited growth to high cell densities of OD₆₀₀ = 20 to 30 (corresponding to 6 to 9 g l^-1 cell dry weight) without the external feed of additional compounds in shake flasks and 96-well plates. The final cell density can be further increased by addition of extra nitrogen during the cultivation. Production of a heterologous triosphosphate isomerase in E. coli BL21(DE3) resulted in 10 times higher volumetric product yield and a higher ratio of soluble to insoluble product when compared to the conventional production method.

Conclusion

The novel EnBase method is robust and simple-to-apply for high cell density cultivation in shake flasks and microwell plates. The potential of the system is that the microbial growth rate and oxygen consumption can be simply controlled by the amount (and principally also by the activity) of the starch-degrading enzyme. This solves the problems of uncontrolled growth, oxygen limitation, and severe pH drop in shaken cultures. In parallel the method provides the basis for enhanced cell densities. The feasibility of the new method has been shown for 96-well plates and shake flasks and we believe that it can easily be adapted to different microwell and deepwell plate formats and shake flasks. Therefore EnBase will be a helpful tool especially in high throughput applications.     

Unusual intragenomic and interspecific variability of <Emphasis Type="Italic">Geobacillus</Emphasis> 16S-23S rRNA internal transcribed spacers

The aim of this study was to evaluate the inter-and intraspecific as well as intragenomic variability of Geobacillus 16S–23S rRNA internal transcribed spacers without tRNA genes and to compare these sequences with sequences bearing tRNA genes. In this study the structural analysis was performed in a unique way because the length and the sequence of the structural blocks were adjusted to fit the structure of 16S–23S rRNA internal transcribed spacers of five different Geobacillus species. Our study demonstrated the mosaic-like structure of 16S–23S rRNA internal transcribed spacers in Geobacillus. Some characteristics of these spacers of geobacilli were not previously reported for other bacteria: unusually short conserved sequence in the 5′ end region, some identical conserved blocks in both 5′ and 3′ regions of 16S–23S rRNA internal transcribed spacers, the same sequence blocks in both 16S–23S and 23S–5S rRNA intergenic spacers. Our study demonstrated quite uniform arrangement of the sequence blocks in Geobacillus thermodenitrificans. This species diverged early in the phylogenetic tree of the genus Geobacillus. For the phylogenetically recent species Geobacillus kaustophilus and Geobacillus lituanicus the low inter-and intraspecific, but high intragenomic variability, as a consequence of recent phylogenetic events, was established.     

Synthesis of half-mustard combi-molecules with fluorescence properties: correlation with EGFR status

The synthesis of 6-(2-chloroethylamino)-4-anilinoquinazolines ZR2002 and ZR2003 designed to block EGFR tyrosine kinase and to damage genomic DNA is described. These compounds present fluorescence properties that permitted the quantitation of their subcellular uptake by flow cytometry. Fluorescence intensities increased with increasing levels of EGFR in a panel of isogenic and established cell lines.     

Novel approach of high cell density recombinant bioprocess development: Optimisation and scale-up from microlitre to pilot scales while maintaining the fed-batch cultivation mode of E. coli cultures

Background

Bioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale.

Results

Here, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase^® technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor.

Conclusions

Cultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditons from the first screening phase not only shortens the developmental line by guarantying that the selected conditions are relevant for the scale up, but in our case also standard batch cultures failed to select the right clone or conditions at all.     

Variation in the contents of pseudohypericin and hypericin in Hypericum perforatum from Lithuania

Hypericin and hypericin-like substances are considered the main active compounds in Hypericum perforatum L. (Hypericaceae). In this work pseudohypericin and hypericin of H. perforatum collected in Lithuania were quantified. Studies on accumulation dynamics and between-accession variation of the contents of these secondary metabolites were carried out by high performance liquid chromatography (HPLC). The data were statistically processed with ANOVA and PCA. Significant difference between pseudohypericin and hypericin content in floral budding and full flowering stages was detected. The highest amounts of the secondary metabolites were observed in the flowering stage. The study revealed evident within population variations in H. perforatum. Mean concentrations of pseudohypericin and hypericin among accessions varied from 3.45 to 6.82 mg/g and from 1.17 to 2.59 mg/g, respectively. Accessions of H. perforatum showed remarkable differences in chemical composition depending on the provenance of plants.     

Klebsiella Phage vB_KleM-RaK2 — A Giant Singleton Virus of the Family Myoviridae

At 346 kbp in size, the genome of a jumbo bacteriophage vB_KleM-RaK2 (RaK2) is the largest Klebsiella infecting myovirus genome sequenced to date. In total, 272 out of 534 RaK2 ORFs lack detectable database homologues. Based on the similarity to biologically defined proteins and/or MS/MS analysis, 117 of RaK2 ORFs were given a functional annotation, including 28 RaK2 ORFs coding for structural proteins that have no reliable homologues to annotated structural proteins in other organisms. The electron micrographs revealed elaborate spike-like structures on the tail fibers of Rak2, suggesting that this phage is an atypical myovirus. While head and tail proteins of RaK2 are mostly myoviridae-related, the bioinformatics analysis indicate that tail fibers/spikes of this phage are formed from podovirus-like peptides predominantly. Overall, these results provide evidence that bacteriophage RaK2 differs profoundly from previously studied viruses of the Myoviridae family.