Holistic differential analysis of embryo-induced alterations in the proteome of bovine endometrium in the preattachment period

During the peri-implantation period, molecular signaling between embryo and endometrium (layer of tissue lining the uterus lumen) is supposed to be crucial for the maintenance of pregnancy. To investigate embryo-induced alterations in the proteome of bovine endometrium in the preattachment period (day 18), we used monozygotic cattle twins (generated by embryo splitting) as a model eliminating genetic variability as a source for proteome differences. One of the twins was pregnant after the transfer of two in vitro produced blastocysts, while the corresponding twin received a sham-transfer and served as a nonpregnant control. The two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of the endometrium samples of three twin pairs (pregnant/nonpregnant) revealed four proteins with significantly higher abundance (p < 10(-9)) in each sample derived from the pregnant animals: Rho GDP dissociation inhibitor beta; 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD); soluble NADP(+)-dependent isocitrate dehydrogenase 1; and acyl-CoA-binding protein. To verify the accuracy of the 2-D DIGE quantification, the abundances of 20 alpha-HSD were quantified by a targeted cleavable isotope-coded affinity tag (ICAT) approach. The mass spectrometry-based ICAT quantification matched perfectly the results obtained by 2-D DIGE quantification, demonstrating the accuracy of our data. These results demonstrate that our model (monozygotic twins) in combination with the appropriate analytical tools is particularly suitable for the detection of the proteins involved in the embryo-maternal interactions.     

Re-infestation of houses by Triatoma dimidiata after intra-domicile insecticide application in the Yucatán peninsula, Mexico

In most countries, Chagas disease transmission control remains based on domestic insecticide application. We thus evaluated the efficacy of intra-domicile cyfluthrin spraying for the control of Triatoma dimidiata, the only Chagas disease vector in the Yucatán peninsula, Mexico, and monitored potential re-infestation every 15 days for up to 9 months. We found that there was a re-infestation of houses by adult bugs starting 4 months after insecticide application, possibly from sylvatic/peridomicile areas. This points out the need to take into account the potential dispersal of sylvatic/peridomestic adult bugs into the domiciles as well as continuity action for an effective vector control.     

Waterborne pathogen detection by use of oligonucleotide-based microarrays

A small-oligonucleotide microarray prototype was designed with probes specific for the universal 16S rRNA and cpn60 genes of several pathogens that are usually encountered in wastewaters. In addition to these two targets, wecE-specific oligonucleotide probes were included in the microarray to enhance its discriminating power within the Enterobacteriaceae family. Universal PCR primers were used to amplify variable regions of 16S rRNA, cpn60, and wecE genes directly in Escherichia coli and Salmonella enterica serovar Typhimurium genomic DNA mixtures (binary); E. coli, S. enterica serovar Typhimurium, and Yersinia enterocolitica genomic DNA mixtures (ternary); or wastewater total DNA. Amplified products were fluorescently labeled and hybridized on the prototype chip. The detection sensitivity for S. enterica serovar Typhimurium was estimated to be on the order of 0.1% (10(4) S. enterica genomes) of the total DNA for the combination of PCR followed by microarray hybridization. The sensitivity of the prototype could be increased by hybridizing amplicons generated by PCR targeting genes specific for a bacterial subgroup, such as wecE genes, instead of universal taxonomic amplicons. However, there was evidence of PCR bias affecting the detection limits of a given pathogen as increasing amounts of a different pathogen were spiked into the test samples. These results demonstrate the feasibility of using DNA microarrays in the detection of waterborne pathogens within mixed populations but also raise the problem of PCR bias in such experiments.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

Hexon Modification to Improve the Activity of Oncolytic Adenovirus Vectors against Neoplastic and Stromal Cells in Pancreatic Cancer

Primary pancreatic carcinoma has an unfavourable prognosis and standard treatment strategies mostly fail in advanced cases. Virotherapy might overcome this resistance to current treatment modalities. However, data from clinical studies with oncolytic viruses, including replicating adenoviral (Ad) vectors, have shown only limited activity against pancreatic cancer and other carcinomas. Since pancreatic carcinomas have a complex tumor architecture and frequently a strong stromal compartment consisting of non-neoplastic cell types (mainly pancreatic stellate cells = hPSCs) and extracellular matrix, it is not surprising that Ad vectors replicating in neoplastic cells will likely fail to eradicate this aggressive tumor type. Because the TGFβ receptor (TGFBR) is expressed on both neoplastic cells and hPSCs we inserted the TGFBR targeting peptide CKS17 into the hypervariable region 5 (HVR5) of the capsid protein hexon with the aim to generate a replicating Ad vector with improved activity in complex tumors. We demonstrated increased transduction of both pancreatic cancer cell lines and of hPSCs and enhanced cytotoxicity in co-cultures of both cell types. Surface plasmon resonance analysis demonstrated decreased binding of coagulation factor X to CKS17-modified Ad particles and in vivo biodistribution studies performed in mice indicated decreased transduction of hepatocytes. Thus, to increase activity of replicating Ad vectors we propose to relax tumor cell selectivity by genetic hexon-mediated targeting to the TGFBR (or other receptors present on both neoplastic and non-neoplastic cells within the tumor) to enable replication also in the stromal cell compartment of tumors, while abolishing hepatocyte transduction, and thereby increasing safety.     

Steroid radiommuoassay: contribution of standards to blank values

The sensitivity of the radioimmunoassay of steroids is considerably reduced by high blank values which may be derived in part from co-chromatographed standards. Blank levels approach the detection limit of the radioimmunoassay of aldosterone, testosterone and androstenedione when 10,000 dpm (30–35 pg) labeled steroids are used as reference standard. When 20 μg aldosterone, testosterone, or androstenedione is used as standard, blank levels of up to 12,800 pg were measured in the radioimmunoassay. Application of the standards on a separate strip does not improve the results. From our experiments it seems clear that contamination is transported by the solvent.     

Feinstruktur der Protonephridien von Paromalostomum proceracauda (Plathelminthes,Macrostomida)

Zusammenfassung Die Protonephridien von Paromalostomum proceracauda bestehen aus je einem Terminalkomplex und einem ausleitenden Kanal. Jeder Terminalkomplex setzt sich aus drei multiciliären Terminalzellen mit jeweils separatem Filtrationsapparat zusammen; die Zellen sind gestaffelt hintereinander angeordnet und bilden ein gemeinsames Reusenlumen. Der Nephridialkanal, der nicht an der Ultrafiltration, sondern nur an Resorptionsvorgängen beteiligt ist, besteht aus mindestens zwei röhrenförmigen, hintereinander liegenden ciliären Zellen. Die jeweils letzte Kanalzelle bildet auch den Nephridialporus. Proximal sind die Protonephridien bis zur Basis der Epidermis vollständig von einer interzellulären Matrix umhüllt.

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Fine structure of the protonephridia of Paromalostomum proceracauda (Plathelminthes, Macrostomida)

Summary The protonephridia of Paromalostomum proceracaud consist, respectively, of a terminal complex and a draining canal. Each terminal complex is composed of three multiciliary terminal cells with separate filtration apparatuses; the cells are staggered, forming a joint basket lumen. The nephridial canal consists of two or more tube-shaped ciliary cells, which are arranged in series; these cells do not participate in ultrafiltration but in resorption processes. The last canal cell also forms the nephroporus. Up to the epidermis, the protonephridia are proximally surrounded by an intercellular matrix.

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Abkürzungen cw Cilienwurzel - ep Epidermiszelle - i Interzellularsubstanz - k Kanalzelle - kl Kanallumen - l Leptotrichien - m Muskulatur des Hautmuskelschlauches - n Kern einer Terminaloder Kanalzelle - r Reusenapparat - rl Reusenlumen - rs Reusenstab - t1, 2, 3 Terminalzelle 1, 2, 3 - v Vakuole     

Functional characterization of the Staphylococcus carnosus SecA protein in Escherichia coli and Bacillus subtilissecA mutant strains

Abstract The cell wall of Candida albicans contains mannoproteins that are covalently associated with β-1,6-glucan. When spheroplasts were allowed to regenerate a new cell wall, initially non-glucosylated cell wall proteins accumulated in the medium. While the spheroplasts became osmotically stable, β-1,6-glucosylated proteins could be identified in their cell wall by SDS-extraction or β-1,3-glucanase digestion. At later stages of regeneration, β-1,3-glucosylated proteins were also found. Hence, incorporation of proteins into the cell wall is accompanied by extracellular coupling to β-1,6-/β-l,3-glucan. The SDS-extractable glucosylated proteins probably represent degradation products of wall proteins rather than their precursors. Tunicamycin delayed, but did not prevent the formation of β-1,6-glucosylated proteins, demonstrating that β-1,6-glucan is not attached to N -glycosidic side-chains of wall proteins.     

The dependency of the size-growth relationship of Norway spruce (Picea abies [L.] Karst.) and European beech (Fagus sylvatica [L.]) in forest stands on long-term site conditions, drought events, and ozone stress

Against a backdrop of increasing climate change, the effects of site conditions, drought events and ozone stress on the size-growth relationship in Norway spruce (Picea abies [L.] Karst.) and European beech (Fagus sylvatica [L.]) stands are analyzed. The size-growth relationship is represented by a straight line defined by intercept and slope of a simple linear equation with stem diameter at height 1.30 m as independent variable and annual stem diameter increment at height 1.30 as dependent variable. On the basis of 64 long-term experimental plots dating back to 1871 and representing an ecological gradient from fertile to poor sites, it is shown that poorer sites exhibit shallower slopes of the linear size-growth relationships than fertile sites. Annual measurements of the size-growth relationship, including the extremely dry years of 1976 and 2003, also showed that lower stand growth rates result in shallower size-growth relationship slopes. By comparing stands with and without experimental twice-ambient ozone exposure between 2000 and 2007, it was found that ozone stress can significantly reduce the slope of the size-growth relationship. This indicates that limiting site condition, whether acute or chronic in nature, distinctly reduces the superiority of tall trees, and that a lower degree of resource limitation increases the steepness of the size-growth relationship. The causes for this behavior and the consequences for stand dynamics, silvicultural treatment and prognostication by models are discussed.