Reexamination In Vitro

Purpose. To introduce additional methods to detect and to quantify single pathogens in the complex biofilm formation on an antibacterial dental material. Materials and Methods. A conventional (ST) and an antibacterial dental composite (B) were manufactured. In vitro: specimens were incubated with a mixture of early colonizers. Bacterial adhesion was analyzed by TaqMan PCR after 8/24 h. In situ: TaqMan PCR and 16S rRNA Next Generation Sequencing (NGS) were performed. Results. In vitro: after 8 h incubation, B was covered by 58.6% of the bacterial amount that was attached to ST. After 24 h, the amount of attached bacteria to ST remained constant on ST only slightly lower on B. In situ: after 8 h the amount of adhering A. viscosus and S. mitis was prominent on ST and reduced on B. NGS revealed that S. sanguinis, S. parasanguinis, and Gemella sanguinis were the mainly attached species with S. sanguinis dominant on ST and S. parasanguinis and G. sanguinis dominant on B. Conclusions. Initial biofilm formation was altered by B. A shift between actinomycetes and streptococci was observed in situ. TaqMan PCR and 16S rRNA NGS revealed comparable results in situ and demonstrated the usefulness of NGS to characterize complex bacterial communities

Abl depletion via autophagy mediates the beneficial effects of quercetin against Alzheimer pathology across species

Alzheimer's disease is the most common age-associated neurodegenerative disorder and the most frequent form of dementia in our society. Aging is a complex biological process concurrently shaped by genetic, dietary and environmental factors and natural compounds are emerging for their beneficial effects against age-related disorders. Besides their antioxidant activity often described in simple model organisms, the molecular mechanisms underlying the beneficial effects of different dietary compounds remain however largely unknown. In the present study, we exploit the nematode Caenorhabditis elegans as a widely established model for aging studies, to test the effects of different natural compounds in vivo and focused on mechanistic aspects of one of them, quercetin, using complementary systems and assays. We show that quercetin has evolutionarily conserved beneficial effects against Alzheimer's disease (AD) pathology: it prevents Amyloid beta (A beta)-induced detrimental effects in different C. elegans AD models and it reduces A beta-secretion in mammalian cells. Mechanistically, we found that the beneficial effects of quercetin are mediated by autophagy-dependent reduced expression of Abl tyrosine kinase. In turn, autophagy is required upon Abl suppression to mediate quercetin's protective effects against A beta toxicity. Our data support the power of C. elegans as an in vivo model to investigate therapeutic options for AD

The full-ORF clone resource of the German cDNA Consortium

<p>Abstract</p> <p>Background</p> <p>With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes.</p> <p>Results</p> <p>Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned.</p> <p>Conclusion</p> <p>The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community.</p

A fast and efficient translational control system for conditional expression of yeast genes

A new artificial regulatory system for essential genes in yeast is described. It prevents translation of target mRNAs upon tetracycline (tc) binding to aptamers introduced into their 5'UTRs. Exploiting direct RNA–ligand interaction renders auxiliary protein factors unnecessary. Therefore, our approach is strain independent and not susceptible to interferences by heterologous expressed regulatory proteins. We use a simple PCR-based strategy, which allows easy tagging of any target gene and the level of gene expression can be adjusted due to various tc aptamer-regulated promoters. As proof of concept, five differently expressed genes were targeted, two of which could not be regulated previously. In all cases, adding tc completely prevented growth and, as shown for Nop14p, rapidly abolished de novo protein synthesis providing a powerful tool for conditional regulation of yeast gene expression

Trehalose-6-phosphate-mediated toxicity determines essentiality of OtsB2 in Mycobacterium tuberculosis in vitro and in mice

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors

Validation of a novel <i>Mho</i> microarray for a comprehensive characterisation of the <i>Mycoplasma hominis</i> action in HeLa cell infection

<div><p><i>Mycoplasma hominis</i> is the second smallest facultative pathogen of the human urogenital tract. With less than 600 protein-encoding genes, it represents an ideal model organism for the study of host-pathogen interactions. For a comprehensive characterisation of the <i>M</i>. <i>hominis</i> action in infection a customized Mho microarray, which was based on two genome sequences (PG21 and LBD-4), was designed to analyze the dynamics of the mycoplasma transcriptome during infection and validated for <i>M</i>. <i>hominis</i> strain FBG. RNA preparation was evaluated and adapted to ensure the highest recovery of mycoplasmal mRNAs from <i>in vitro</i> HeLa cell infection assays. Following cRNA hybridization, the read-out strategy of the hybridization results was optimized and confirmed by RT-PCR. A statistically robust infection assay with <i>M</i>. <i>hominis</i> strain FBG enabled the identification of differentially regulated key effector molecules such as critical cytoadhesins (4 h post infection (pI)), invasins (48 h pI) and proteins associated with establishing chronic infection of the host (336 h pI). Of the 294 differentially regulated genes (>2-fold) 128 (43.5%) encoded hypothetical proteins, including lipoproteins that seem to play a central role as virulence factors at each stage of infection: P75 as a novel cytoadhesin candidate, which is also differentially upregulated in chronic infection; the MHO_2100 protein, a postulated invasin and the MHO_730-protein, a novel <i>ecto</i>-nuclease and domain of an ABC transporter, the function of which in chronic infection has still to be elucidated. Implementation of the <i>M</i>. <i>hominis</i> microarray strategy led to a comprehensive identification of to date unknown candidates for virulence factors at relevant stages of host cell infection.</p></div

Comparison of microarray and RT-qPCR results.

<p>Total RNA of <i>M</i>. <i>hominis</i>-infected HeLa cells for 4 h, 48 h or 336 h was subjected to Mho-microarray or RT-qPCR analyses and the change in expression levels of the named genes, with respect to that at the start of infection (1 h for RNAs of 08/15 and 0 h for RNAs of 04/11 and 11/11), quantified as described in the Method section.</p