Peptidyl-Prolyl-cis/trans-Isomerases Mip and PpiB of Legionella pneumophila Contribute to Surface Translocation, Growth at Suboptimal Temperature, and Infection.

The gammaproteobacterium Legionella pneumophila is the causative agent of Legionnaires' disease, an atypical pneumonia that manifests itself with severe lung damage. L. pneumophila, a common inhabitant of freshwater environments, replicates in free-living amoebae and persists in biofilms in natural and man-made water systems. Its environmental versatility is reflected in its ability to survive and grow within a broad temperature range as well as its capability to colonize and infect a wide range of hosts, including protozoa and humans. Peptidyl-prolyl-cis/trans-isomerases (PPIases) are multifunctional proteins that are mainly involved in protein folding and secretion in bacteria. In L. pneumophila the surface-associated PPIase Mip was shown to facilitate the establishment of the intracellular infection cycle in its early stages. The cytoplasmic PpiB was shown to promote cold tolerance. Here, we set out to analyze the interrelationship of these two relevant PPIases in the context of environmental fitness and infection. We demonstrate that the PPIases Mip and PpiB are important for surfactant-dependent sliding motility and adaptation to suboptimal temperatures, features that contribute to the environmental fitness of L. pneumophila Furthermore, they contribute to infection of the natural host Acanthamoeba castellanii as well as human macrophages and human explanted lung tissue. These effects were additive in the case of sliding motility or synergistic in the case of temperature tolerance and infection, as assessed by the behavior of the double mutant. Accordingly, we propose that Mip and PpiB are virulence modulators of L. pneumophila with compensatory action and pleiotropic effects.

Image formation properties and inverse imaging problem in aperture based scanning near field optical microscopy.

Aperture based scanning near field optical microscopes are important instruments to study light at the nanoscale and to understand the optical functionality of photonic nanostructures. In general, a detected image is affected by both the transverse electric and magnetic field components of light. The discrimination of the individual field components is challenging as these four field components are contained within two signals in the case of a polarization resolved measurement. Here, we develop a methodology to solve the inverse imaging problem and to retrieve the vectorial field components from polarization and phase resolved measurements. Our methodology relies on the discussion of the image formation process in aperture based scanning near field optical microscopes. On this basis, we are also able to explain how the relative contributions of the electric and magnetic field components within detected images depend on the chosen probe. We can therefore also describe the influence of geometrical and material parameters of individual probes within the image formation process. This allows probes to be designed that are primarily sensitive either to the electric or magnetic field components of light.

High speed and high resolution table-top nanoscale imaging.

We present a table-top coherent diffractive imaging (CDI) experiment based on high-order harmonics generated at 18 nm by a high average power femtosecond fiber laser system. The high photon flux, narrow spectral bandwidth, and high degree of spatial coherence allow for ultrahigh subwavelength resolution imaging at a high numerical aperture. Our experiments demonstrate a half-pitch resolution of 15 nm, close to the actual Abbe limit of 12 nm, which is the highest resolution achieved from any table-top extreme ultraviolet (XUV) or x-ray microscope. In addition, sub-30 nm resolution was achieved with only 3 s of integration time, bringing live diffractive imaging and three-dimensional tomography on the nanoscale one step closer to reality. The current resolution is solely limited by the wavelength and the detector size. Thus, table-top nanoscopes with only a few-nanometer resolutions are in reach and will find applications in many areas of science and technology.

[VATS lobectomy--a standard procedure in the therapy for stage I non-small cell lung cancer?]. / VATS-Lobektomie--Ein Standardverfahren in der Therapie des nicht kleinzelligen Lungenkarzinoms im Stadium I?

Even though VATS lobectomy has been practised since 1991 in stage I of non-small cell lung cancer (NSCLC), it was not being considered equivalent to conventional lobectomy due to considerable doubts in terms of safety and oncological permissibility. This study describes our experience and an evaluation of the systematic establishment of lobectomy by means of video-assisted thoracic surgery (VATS) as standard treatment of NSCLC in stage I, which serves as an alternative to conventional surgery. For this purpose, 42 NSCLC patients in stage I, undergoing a conventional lobectomy in 2010 (group I), were retrospectively compared to 30 patients in the same tumour stage (group II) who were treated in 2011 using VATS lobectomy. The comparison of these two groups was drawn regarding operation time, number of resected lymph nodes, required analgesics, duration of drainage, rate of postoperative complications and length of hospital stay. Although VATS lobectomy requires a longer operation time of approximately 30 minutes, it shows significant advantages in reference to postoperative need of analgesics, duration of drainage and complications after surgery. Furthermore, the amount of resected lymph nodes was comparable in both groups. Therefore, VATS lobectomy constitutes an essential extension for the operative management in a lung cancer centre. Our results show that this new method is not only of equal, but of superior value compared to conventional lobectomy. Our experience and recent data in the literature illustrate that VATS lobectomy will play a decisive role in therapy for NSCLC in stage I, potentially even in stages II and IIIA.

Effects of non-vascularized adipose tissue transplantation on its genetic profile.

Subcutaneous adipose tissue (SAT) is recognized as a highly active metabolic and inflammatory tissue. Interestingly, adipose tissue transplantation is widely performed in plastic surgery via lipofilling, yet little is known about the gene alteration of adipocytes after transplantation. We performed an RNA-expression analysis of fat transplants before and after fat transplantation.In C57BL/6 N mice SAT was autologously transplanted. Samples of SAT were analysed before transplantation, 7, and 15 days after transplantation and gene expression profiles were measured.Analysis revealed that lipid metabolism-related genes were downregulated while inflammatory and extracellular matrix related genes were up-regulated 7 and 15 days after transplantation. When comparing gene expression profile 7 days after transplantation to 15 days after transplantation developmental pathways showed most changes.

[Physical lipolysis]. / Physikalische Lipolyse.

The number of non-invasive procedures is steadily growing. Lipolysis with focused ultrasound, cryo-lipolysis, laser-lipolysis, radiofrequency-lipolysis, HIFU-lipolysis, classic liposuction, vibration-assisted liposuction and water beam-assisted Liposuction are newly established procedures to treat diet- and sport resistant fat deposits. Non-invasive lipolysis with focused ultrasound in combination with radiofrequency as well as cryo-lipolysis are the most established procedures. The methods are described in detail and evaluated.

The loss of kinetoplastic DNA in two species of Trypanosomatidae treated with acriflavine.

The effects of acriflavine on two species of Trypanosomatidae, Crithidia luciliae and Trypanosoma mega, have been investigated. It has been observed that kinetoplastic (i.e. mitochondrial) DNA is lost in a high percentage of acriflavine-treated cells. Resting flagellates, from stationary-phase or hemin-deficient cultures, are considerably more resistant to the acridine than are flagellates from a log-phase culture. When the kinetoplast has retained some DNA and still remains visible in stained smears, it appears reduced in size, and its ultrastructure is extremely abnormal: the DNA fibrils, clearly visible in normal kinetoplasts, are condensed; they appear as an electron-opaque, apparently homogeneous mass, separated from the membranes by a space of low electron-opacity. Analyses of DNA extracts, with high speed centrifugation in CsCl density gradients, revealed that the satellite band, presumably kinetoplastic DNA, is lost by trypanosomes grown for 5 days in the presence of acriflavine. Radioautography was used to study the effects of acriflavine on thymidine-(3)H incorporation in C. luciliae. At the concentration which affects the kinetoplast specifically, the dye produces an 87% inhibition of thymidine incorporation in this organelle. The kinetics of this inhibition suggest a direct effect on replication. No decrease in incorporation occurs in the nucleus. These results lead to the conclusion that loss of kinetoplastic DNA is due to continued growth and cell division in the absence of kinetoplastic DNA replication. Several hypotheses are discussed concerning the specificity of the dye's action upon the replication of extrachromosomal DNA.

High resolution XUV Fourier transform holography on a table top.

Today, coherent imaging techniques provide the highest resolution in the extreme ultraviolet (XUV) and X-ray regions. Fourier transform holography (FTH) is particularly unique, providing robust and straightforward image reconstruction at the same time. Here, we combine two important advances: First, our experiment is based on a table-top light source which is compact, scalable and highly accessible. Second, we demonstrate the highest resolution ever achieved with FTH at any light source (34 nm) by utilizing a high photon flux source and cutting-edge nanofabrication technology. The performance, versatility and reliability of our approach allows imaging of complex wavelength-scale structures, including wave guiding effects within these structures, and resolving embedded nanoscale features, which are invisible for electron microscopes. Our work represents an important step towards real-world applications and a broad use of XUV imaging in many areas of science and technology. Even nanoscale studies of ultra-fast dynamics are within reach.

Dissection of antigenic and irritative effects of epicutaneously applied haptens in mice. Evidence that not the antigenic component but nonspecific proinflammatory effects of haptens determine the concentration-dependent elicitation of allergic contact dermatitis.

Allergic contact dermatitis differs from most other immune reactions by its strict dose dependence during the elicitation phase. Moreover, almost all known contact allergens can also induce dose-dependent irritative dermatitis and in general only elicit allergic contact dermatitis in sensitized individuals when applied within a narrow dose range. Therefore, we hypothesized that elicitation of contact hypersensitivity (CHS) may require two signals, antigen-specific effector cell activation and a non-antigen-specific proinflammatory signal, both of which are provided by application of a sufficient dose of hapten. To dissociate these putative two signals, oxazolone-sensitized mice were ear challenged with a dose of the specific hapten which was too low to elicit CHS. At the same time, an unrelated hapten was applied in a conventional concentration to the same skin site. Whereas neither treatment alone elicited a significant CHS response, application of both compounds together resulted in a strong CHS response that was indistinguishable from that elicited by the full dose of the specific hapten. Upon coadministration of the irrelevant hapten, allergic contact dermatitis could be elicited even when the dose of the specific hapten was further reduced by a factor of 10(3). In contrast, a dose reduction of the irrelevant hapten by a factor of two resulted in the loss of the CRS response. These data indicate that non-antigen-specific effects of epicutaneously applied haptens significantly contribute to the elicitation of CHS responses and that the capacity of the hapten to evoke this proinflammatory stimulus rather than its antigenicity is responsible for the strict concentration dependence.

Trypanosoma brucei: posttranscriptional control of the variable surface glycoprotein gene expression site.

The arrest of variable surface glycoprotein (VSG) synthesis is one of the first events accompanying the differentiation of Trypanosoma brucei bloodstream forms into procyclic forms, which are characteristic of the insect vector. This is because of a very fast inhibition of VSG gene transcription which occurs as soon as the temperature is lowered. We report that this effect is probably not controlled at the level of transcription initiation, since the beginning of the VSG gene expression site, about 45 kilobases upstream from the antigen gene, remains transcribed in procyclic forms. The permanent activity of the promoter readily accounts for the systematic reappearance, upon return to the bloodstream form after cyclical transmission, of the antigen type present before passage to the tsetse fly. The abortive transcription of the VSG gene expression site appears linked to RNA processing abnormalities. Such posttranscriptional controls may allow the modulation of gene expression in a genome organized in large multigenic transcription units.

Trypanosoma brucei: enrichment by UV of intergenic transcripts from the variable surface glycoprotein gene expression site.

The expression site for the variable surface glycoprotein (VSG) gene AnTat 1.3A of Trypanosoma brucei is 45 kilobases long and encompasses seven expression site-associated genes (ESAGs) (E. Pays, P. Tebabi, A. Pays, H. Coquelet, P. Revelard, D. Salmon, and M. Steinert, Cell 57:835-845, 1989). After UV irradiation, several large transcripts from the putative promoter region were strongly enriched. We report that one such major transcript starts near the poly(A) addition site of the first gene (ESAG 7), spans the intergenic region, and extends to the poly(A) addition site of the second gene (ESAG 6), thus bypassing the normal 3' splice site of the ESAG 6 mRNA. Since this transcript is spliced, we conclude that UV irradiation does not inhibit splicing but stabilizes unstable processing products. This demonstrates that at least some intergenic regions of the VSG gene expression site are continuously transcribed in accordance with a polycistronic transcription model.

Structure and transcription of the actin gene of Trypanosoma brucei.

In Trypanosoma brucei, the actin gene is present in a cluster of two, three, or four tandemly linked copies, depending on the strain. Each cluster seems to exist in two allelic versions, as suggested by the polymorphism of both gene number and restriction fragment length in the DNA from cloned trypanosomes. The amplification of the gene copy number probably occurs through unequal sister chromatid exchange. The chromosomes harboring the actin genes belong to the large size class. The coding sequence was 1,128 nucleotides long and showed 60 to 70% homology to other eucaryotic actin genes. Surprisingly, this homology seemed weaker with Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma vivax, Trypanosoma mega, or Leishmania actin-specific sequences. The mRNA was around 1.6 kilobases long and was synthesized at the same level in bloodstream and procyclic forms of the parasite. Large RNA precursors, up to 7.7 kilobases, were found in a pattern identical in strains containing either two or three gene copies. Probing of the flanking regions of the gene with either steady-state or in vitro transcripts, as well as S1 nuclease protection and primer extension experiments, allowed mapping of the 3' splice site of the actin mRNA, 38 nucleotides upstream from the translation initiation codon. A variably sized poly(dT) tract was found about 30 base pairs ahead of the splice site. The largest detected actin mRNA precursor seemed to give rise to at least two additional stable mRNAs. The RNA polymerase transcribing the actin gene exhibited the same sensitivity to inhibition by alpha-amanitin as that transcribing both the spliced leader and the bulk of polyadenylated mRNAs.

Putative genes of a variant-specific antigen gene transcription unit in Trypanosoma brucei.

In a 7-kilobase (kb) sequence upstream from the 5' barren region, the Trypanosoma brucei AnTat 1.3A expression site carries two putative genes, named ESAG 2 and ESAG 3 for expression site-associated genes, as well as a copy of ESAG 1 (D.F. Cully, H.S. Ip, and G.A.M. Cross, Cell 42:173-182, 1985). At least 3 kb of this expression site exhibits a high degree of homology with the silent telomere carrying the AnTat 1.3A basic copy, whose ESAG 1 is interrupted by stop codons. Like the antigen gene, the region containing the ESAGs is transcribed only in the bloodstream forms, although transcription of 5' barren- and ESAG 2-related sequences also occurs in cultured procyclics. Analysis of steady-state and nascent transcripts suggests a continuous transcription of the whole expression site by an RNA polymerase resistant to alpha-amanitin, possibly initiating at a polymerase I-like promoter located about 17 kb upstream from the antigen gene. This polymerase seems prone to becoming inactivated upon incubation of the trypanosomes at low temperature. The putative protein encoded by ESAG 3 may carry a hydrophobic signal peptide, suggesting interaction with a membrane.

Isolation of Staphylococcus sciuri from horse skin infection.

Staphylococcus sciuri is known as an opportunistic pathogen colonizing domesticated animals and has also been associated with wound infections in humans. Particularly over the last decade, oxacillin (methicillin) resistant strains had been emerged, which now increase the medical relevance of this species. This report describes the identification of an oxacillin-resistant S. sciuri isolate from a wound infection of a horse. We determined the absence of coagulase and hyaluronidase activity and analysed the antibiotic resistance profile.

Common molecular mechanisms of symbiosis and pathogenesis.

Traditionally, symbiotic and pathogenic interactions were considered different manifestations of the bacteria-host interaction. However, the molecular mechanisms that mediate communication between and cellular modulation of the involved partners are quite similar. With this review we aim to contribute to a reduction of the traditional gap between symbiosis and pathogenesis research.

Characterization of long and short repetitive sequences in the sea urchin genome.

Long and short repetitive sequences were purified from the DNA of Paracentrotus lividus under conditions designed to optimize the yield of complete, end to end sequences. Double-stranded long repeat DNA prepared in this manner ranged in length from approximately 3000 to 15 000 nucleotide pairs with average sizes of approximately 6000 base pairs. In the electron microscope, long repeat DNA was observed to possess continuous sequences that often appeared to be terminated by one or more loops and/or fold backs. Long repeat DNA sequences, resheared to 300 base pairs, were found to have an average melting point identical to that for sheared native DNA. Thus, the reassociated duplexes of long repetitive DNA seem to possess very few mismatched base pairs. Reassociation kinetic analyses indicate that the majority of the long repeat sequences are reiterated only 4--7 times per haploid amount of DNA. Melt-reassociation analyses of short repetitive DNA, at several criteria, support the previously held concept that these sequences belong the sets or families of sequences which are inexact copies of one another. Our studies also support hypotheses suggesting that short repetitive sequences belong to families which may have arisen via distinct salttatory events. The relationships between long and short repetitive DNA sequences are considered with respect to widely held concepts of their sequence organization, evolution, and possible functions within eucaryotic genomes. A model for the possible organization of short repeats within long repetitive DNA sequences is also presented.

Trypanosoma brucei repeated element with unusual structural and transcriptional properties.

The genome of Trypanosoma brucei contains up to 400 copies of a conserved sequence (TRS, trypanosome repeated sequence). The majority of TRS copies (TRS1) are 5.2 X 10(3) base-pairs (kb) and are flanked by different separate halves of the previously described transposable element RIME (ribosomal mobile element), although a variant copy (TRS2) contains only the central 1.45 kb portion and lacks RIME. TRS1 elements can probably undergo transposition, since they are dispersed in all chromosome size classes and are bordered by direct repeats of about four base-pairs. Some TRS1 elements may contain an open reading frame over almost their entire length (1651 codons), encoding a protein showing homology with reverse transcriptase. TRS probes detect poly(A)+ transcripts of 5 to 9 kb, generated by a polymerase moderately sensitive to alpha-amanitin. Transcription is developmentally regulated. Both TRS and RIME sense transcripts are preferentially synthesized compared to anti-sense transcripts, and are much more abundant in bloodstream forms than in cultured procyclics.

Enhancing 18F-FDG-PET/CT analysis in lung cancer patients. Is CT-CT image fusion helpful in predicting pleural involvement? A pilot study.

AIM: To retrospectively evaluate the feasibility and value of CT-CT image fusion to assess the shift of peripheral lung cancers with/-out chest wall infiltration, comparing computed tomography acquisitions in shallow-breathing (SB-CT) and deep-inspiration breath-hold (DIBH-CT) in patients undergoing FDG-PET/CT for lung cancer staging. METHODS: Image fusion of SB-CT and DIBH-CT was performed with a multimodal workstation used for nuclear medicine fusion imaging. The distance of intrathoracic landmarks and the positional shift of tumours were measured using semi-transparent overlay of both CT series. Statistical analyses were adjusted for confounders of tumour infiltration. Cutoff levels were calculated for prediction of no-/infiltration. RESULTS: Lateral pleural recessus and diaphragm showed the largest respiratory excursions. Infiltrating lung cancers showed more limited respiratory shifts than non-infiltrating tumours. A large respiratory tumour-motility accurately predicted non-infiltration. However, the tumour shifts were limited and variable, limiting the accuracy of prediction. CONCLUSION: This pilot fusion study proved feasible and allowed a simple analysis of the respiratory shifts of peripheral lung tumours using CT-CT image fusion in a PET/CT setting. The calculated cutoffs were useful in predicting the exclusion of chest wall infiltration but did not accurately predict tumour infiltration. This method can provide additional qualitative information in patients with lung cancers with contact to the chest wall but unclear CT evidence of infiltration undergoing PET/CT without the need of additional investigations. Considering the small sample size investigated, further studies are necessary to verify the obtained results.

Microbial diversity of marine sponges.

The recent application of molecular microbial ecology tools to sponge-microbe associations has revealed a glimpse into the biodiversity of these microbial communities, that is considered just 'the tip of the iceberg'. This chapter provides an overview over these new findings with regard to identity, diversity and distribution patterns of sponge-associated microbial consortia. The sponges Aplysina aerophoba (Verongida), Rhopaloeides odorabile (Dicytoceratida) and Theonella swinhoei (Lithistida) were chosen as model systems for this review because they have been subject to both, cultivation-dependent and cultivation-independent approaches. A discussion of the microbial assemblages of Halichondriapanicea is presented in the accompanying chapter by Imhoff and Stöhr. Considering that a large fraction of sponge-associated microbes is not yet amenable to cultivation, an emphasis has been placed on the techniques centering around the 16S rRNA gene. A section has been included that covers the potential of sponge microbial communities for drug discovery. Finally, a 'sponge-microbe interaction model' is presented that summarizes our current understanding of the processes that might have shaped the community structure of the microbial assemblages within sponges.