Publications by authors named "Gudrun Kohl"

23 Publications

  • Page 1 of 1

An Economical and Flexible Dual Barcoding, Two-Step PCR Approach for Highly Multiplexed Amplicon Sequencing.

Front Microbiol 2021 20;12:669776. Epub 2021 May 20.

Joint Microbiome Facility of the Medical University of Vienna and the University of Vienna, Vienna, Austria.

In microbiome research, phylogenetic and functional marker gene amplicon sequencing is the most commonly-used community profiling approach. Consequently, a plethora of protocols for the preparation and multiplexing of samples for amplicon sequencing have been developed. Here, we present two economical high-throughput gene amplification and sequencing workflows that are implemented as standard operating procedures at the Joint Microbiome Facility of the Medical University of Vienna and the University of Vienna. These workflows are based on a previously-published two-step PCR approach, but have been updated to either increase the accuracy of results, or alternatively to achieve orders of magnitude higher numbers of samples to be multiplexed in a single sequencing run. The high-accuracy workflow relies on unique dual sample barcoding. It allows the same level of sample multiplexing as the previously-published two-step PCR approach, but effectively eliminates residual read missasignments between samples (crosstalk) which are inherent to single barcoding approaches. The high-multiplexing workflow is based on combinatorial dual sample barcoding, which theoretically allows for multiplexing up to 299,756 amplicon libraries of the same target gene in a single massively-parallelized amplicon sequencing run. Both workflows presented here are highly economical, easy to implement, and can, without significant modifications or cost, be applied to any target gene of interest.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2021.669776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173057PMC
May 2021

Combined hormonal contraceptives are associated with minor changes in composition and diversity in gut microbiota of healthy women.

Environ Microbiol 2021 Jun 6;23(6):3037-3047. Epub 2021 May 6.

Centre for Microbiology and Environmental Systems Science, Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria.

Recent human and animal studies have found associations between gut microbiota composition and serum levels of sex hormones, indicating that they could be an important factor in shaping the microbiota. However, little is known about the effect of regular hormonal fluctuations over the menstrual cycle or CHC-related changes of hormone levels on gut microbiota structure, diversity and dynamics. The aim of this study was to investigate the effect of CHCs on human gut microbiota composition. The effect of CHC pill intake on gut microbiota composition was studied in a group of seven healthy pre-menopausal women using the CHC pill, compared to the control group of nine age-matched healthy women that have not used hormonal contraceptives in the 6 months prior to the start of the study. By analysing the gut microbiota composition in both groups during one menstrual cycle, we found that CHC usage is associated with a minor decrease in gut microbiota diversity and differences in the abundance of several bacterial taxa. These results call for further investigation of the mechanisms underlying hormonal and hormonal contraceptive-related changes of the gut microbiota and the potential implications of these changes for women's health.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1462-2920.15517DOI Listing
June 2021

Genetic variation in an ephemeral mudflat species: The role of the soil seed bank and dispersal in river and secondary anthropogenic habitats.

Ecol Evol 2020 Apr 17;10(8):3620-3635. Epub 2020 Mar 17.

Department of Integrative Biology and Biodiversity Research Institute of Botany University of Natural Resources and Life Sciences Vienna Austria.

Many ephemeral mudflat species, which rely on a soil seed bank to build up the next generation, are endangered in their natural habitat due to the widespread regulation of rivers. The aim of the present study was to elucidate the role of the soil seed bank and dispersal for the maintenance of genetic diversity in populations of near-natural river habitats and anthropogenic habitats created by traditional fish farming practices using as a model. Using microsatellite markers, we found no difference in genetic diversity levels between soil seed bank and above-ground population and only moderate differentiation between the two fractions. One possible interpretation is the difference in short-term selection during germination under specific conditions (glasshouse versus field) resulting in an ecological filtering of genotypes out of the reservoir in the soil. River populations harbored significantly more genetic diversity than populations from the anthropogenic pond types. We suggest that altered levels and patterns of dispersal together with stronger selection pressures and historical bottlenecks in anthropogenic habitats are responsible for the observed reduction in genetic diversity. Dispersal is also supposed to largely prohibit genetic structure across Europe, although there is a gradient in private allelic richness from southern Europe (high values) to northern, especially north-western, Europe (low values), which probably relates to postglacial expansion out of southern and/or eastern refugia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ece3.6109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160169PMC
April 2020

Proton-sensing G protein-coupled receptors as regulators of cell proliferation and migration during tumor growth and wound healing.

Exp Dermatol 2017 02;26(2):127-132

Department of Dermatology, University Medical Center Regensburg, Regensburg, Germany.

Dysregulation of pH is a feature of both tumor growth and tissue repair. In tumors, microenvironmental changes, like in lactate metabolism, lead to altered intra- and extracellular pH (pH , pH ) and vice versa. In wounds, barrier disruption results in extensive variations in pH on the wound surface. It is known that altered extracellular proton concentrations have a major impact on cell turnover and migration as well as on the metabolic activity of cells involved in tumor spread and wound closure. The proton-sensing G protein-coupled receptors (GPCRs) GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A) are activated via a decrease in pH and transduce this signal to molecular intracellular pathways. Based on the current knowledge, we speculate on the role of proton-sensing GPCRs in wound healing and on their potential as mechanistic linkers of tumor growth and tissue repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/exd.13209DOI Listing
February 2017

IRAK-M Expression in Tumor Cells Supports Colorectal Cancer Progression through Reduction of Antimicrobial Defense and Stabilization of STAT3.

Cancer Cell 2016 05 14;29(5):684-696. Epub 2016 Apr 14.

Department of Surgery, University Medical Center Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany; Regensburg Center for Interventional Immunology, University Medical Center Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany. Electronic address:

Colorectal cancer (CRC) is associated with loss of epithelial barrier integrity, which facilitates the interaction of the immunological microenvironment with the luminal microbiome, eliciting tumor-supportive inflammation. An important regulator of intestinal inflammatory responses is IRAK-M, a negative regulator of TLR signaling. Here we investigate the compartment-specific impact of IRAK-M on colorectal carcinogenesis using a mouse model. We demonstrate that IRAK-M is expressed in tumor cells due to combined TLR and Wnt activation. Tumor cell-intrinsic IRAK-M is responsible for regulation of microbial colonization of tumors and STAT3 protein stability in tumor cells, leading to tumor cell proliferation. IRAK-M expression in human CRCs is associated with poor prognosis. These results suggest that IRAK-M may be a potential therapeutic target for CRC treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccell.2016.03.014DOI Listing
May 2016

Genetic consequences of cladogenetic vs. anagenetic speciation in endemic plants of oceanic islands.

AoB Plants 2015 Aug 26;7. Epub 2015 Aug 26.

Department of Botany and Biodiversity Research, University of Vienna, Rennweg 14, A-1030 Vienna, Austria Herbarium, Department of Evolution, Ecology, and Organismal Biology, The Ohio State University, 1315 Kinnear Road, Columbus, OH 43212, USA

Adaptive radiation is a common mode of speciation among plants endemic to oceanic islands. This pattern is one of cladogenesis, or splitting of the founder population, into diverse lineages in divergent habitats. In contrast, endemic species have also evolved primarily by simple transformations from progenitors in source regions. This is anagenesis, whereby the founding population changes genetically and morphologically over time primarily through mutation and recombination. Gene flow among populations is maintained in a homogeneous environment with no splitting events. Genetic consequences of these modes of speciation have been examined in the Juan Fernández Archipelago, which contains two principal islands of differing geological ages. This article summarizes population genetic results (nearly 4000 analyses) from examination of 15 endemic species, involving 1716 and 1870 individuals in 162 and 163 populations (with amplified fragment length polymorphisms and simple sequence repeats, respectively) in the following genera: Drimys (Winteraceae), Myrceugenia (Myrtaceae), Rhaphithamnus (Verbenaceae), Robinsonia (Asteraceae, Senecioneae) and Erigeron (Asteraceae, Astereae). The results indicate that species originating anagenetically show high levels of genetic variation within the island population and no geographic genetic partitioning. This contrasts with cladogenetic species that show less genetic diversity within and among populations. Species that have been derived anagenetically on the younger island (1-2 Ma) contain less genetic variation than those that have anagenetically speciated on the older island (4 Ma). Genetic distinctness among cladogenetically derived species on the older island is greater than among similarly derived species on the younger island. An important point is that the total genetic variation within each genus analysed is comparable, regardless of whether adaptive divergence occurs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/aobpla/plv102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605995PMC
August 2015

Progressive migration and anagenesis in Drimys confertifolia of the Juan Fernández Archipelago, Chile.

J Plant Res 2015 Jan 8;128(1):73-90. Epub 2014 Oct 8.

Departamento de Botánica, Universidad de Concepción, Casilla 160-C, Concepción, Chile.

A common mode of speciation in oceanic islands is by anagenesis, wherein an immigrant arrives and through time transforms by mutation, recombination, and drift into a morphologically and genetically distinct species, with the new species accumulating a high level of genetic diversity. We investigate speciation in Drimys confertifolia, endemic to the two major islands of the Juan Fernández Archipelago, Chile, to determine genetic consequences of anagenesis, to examine relationships among populations of D. confertifolia and the continental species D. winteri and D. andina, and to test probable migration routes between the major islands. Population genetic analyses were conducted using AFLPs and nuclear microsatellites of 421 individuals from 42 populations from the Juan Fernández islands and the continent. Drimys confertifolia shows a wide genetic variation within populations on both islands, and values of genetic diversity within populations are similar to those found within populations of the continental progenitor. The genetic results are compatible with the hypothesis of high levels of genetic variation accumulating within anagenetically derived species in oceanic islands, and with the concept of little or no geographical partitioning of this variation over the landscape. Analysis of the probability of migration within the archipelago confirms colonization from the older island, Robinson Crusoe, to the younger island Alejandro Selkirk.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10265-014-0666-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4300435PMC
January 2015

Relationships and genetic consequences of contrasting modes of speciation among endemic species of Robinsonia (Asteraceae, Senecioneae) of the Juan Fernández Archipelago, Chile, based on AFLPs and SSRs.

New Phytol 2015 Jan 10;205(1):415-28. Epub 2014 Sep 10.

The University Museum, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-0033, Japan.

This study analyses and compares the genetic signatures of anagenetic and cladogenetic speciation in six species of the genus Robinsonia (Asteraceae, Senecioneae), endemic to the Juan Fernández Islands, Chile. Population genetic structure was analyzed by amplified fragment length polymorphism (AFLP) and microsatellite (simple sequence repeat, SSR) markers from 286 and 320 individuals, respectively, in 28 populations. Each species is genetically distinct. Previous hypotheses of classification among these species into subgenera and sections, via morphological, phytochemical, isozymic and internal transcribed spacer (ITS) data, have been confirmed, except that R. saxatilis appears to be related to R. gayana rather than R. evenia. Analysis of phylogenetic results and biogeographic context suggests that five of these species have originated by cladogenesis and adaptive radiation on the older Robinson Crusoe Island. The sixth species, R. masafuerae, restricted to the younger Alejandro Selkirk Island, is closely related to and an anagenetic derivative of R. evenia from Robinson Crusoe. Microsatellite and AFLP data reveal considerable genetic variation among the cladogenetically derived species of Robinsonia, but within each the genetic variation is lower, highlighting presumptive genetic isolation and rapid radiation. The anagenetically derived R. masafuerae harbors a level of genetic variation similar to that of its progenitor, R. evenia. This is the first direct comparison of the genetic consequences of anagenetic and cladogenetic speciation in plants of an oceanic archipelago.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/nph.13000DOI Listing
January 2015

Genetic variation (AFLPs and nuclear microsatellites) in two anagenetically derived endemic species of Myrceugenia (Myrtaceae) on the Juan Fernández Islands, Chile.

Am J Bot 2013 Apr 18;100(4):722-34. Epub 2013 Mar 18.

Department of Systematic and Evolutionary Botany, Biodiversity Center, University of Vienna, Rennweg 14, A-1030 Vienna, Austria.

Premise Of The Study: Anagenesis (or phyletic evolution) is one mode of speciation that occurs in the evolution of plants on oceanic islands. Of two endemic species on the Juan Fernández Islands (Chile), Myrceugenia fernandeziana and M. schulzei (Myrtaceae), believed to have originated anagenetically from different continental progenitors, the first is endemic to Robinson Crusoe Island and has no clear tie to continental relatives; the last is endemic to the younger island, Alejandro Selkirk Island, and has close affinity to M. colchaguensis in mainland Chile.

Methods: Using AFLPs and six nuclear microsatellites from 381 individuals representing 33 populations, we determined patterns of genetic variation within and among populations on both islands and between those of the islands and mainland.

Key Results: Considerable genetic variation was found within populations on both islands. The level of gene diversity within M. schulzei was equivalent to that of its close continental relative M. colchaguensis. Genetic diversity was not partitioned geographically in M. fernandeziana and was weakly so and nonsignificantly in M. schulzei.

Conclusions: The high genetic variation in both taxa is most likely due to anagenetic speciation. Subsidence of the older island Robinson Crusoe, landscape erosion, and restructuring of communities have severely reduced the overall island population to a single panmictic system. On the younger and less modified Alejandro Selkirk Island, slightly stronger patterns of genetic divergence are seen in M. schulzei. Because both species are genetically diverse and number in the thousands of individuals, neither is presently endangered in the archipelago.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3732/ajb.1200541DOI Listing
April 2013

Development of microsatellite markers in (Asteraceae) an endemic genus of the Juan Fernández Archipelago, Chile.

Conserv Genet Resour 2013 Mar 19;5(1):63-67. Epub 2012 Aug 19.

Department of Systematic and Evolutionary Botany, Biodiversity Center, University of Vienna, Rennweg 14, 1030 Vienna, Austria.

Ten microsatellite markers were developed for (Asteraceae), a genus endemic to the Juan Fernández Archipelago, Chile. Polymorphisms of these markers were tested using one population each of , , and . The number of alleles for these markers ranged from 2 to 17 per locus, and expected heterozygosity ranged from 0 to 0.847 by population. A significant deviation from Hardy-Weinberg equilibrium was observed in zero to two markers in each population, and no significant linkage disequilibrium between markers was detected. The markers reported here would be useful for evolutionary studies and conservation strategies in .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12686-012-9734-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3579811PMC
March 2013

Development of microsatellite markers in species of Erigeron (Asteraceae) endemic to the Juan Fernández Archipelago, Chile.

Am J Bot 2012 Dec 28;99(12):e487-9. Epub 2012 Nov 28.

Department of Systematic and Evolutionary Botany, Biodiversity Center, University of Vienna, Rennweg 14, A-1030 Vienna, Austria.

Unlabelled:

Premise Of The Study: Microsatellite markers were developed in Erigeron rupicola and tested by amplification in six Erigeron species endemic to the Juan Fernández Archipelago, Chile, to investigate genetic diversity and population structure. •

Methods And Results: Using 454 pyrosequencing, 24 primer pairs were developed in E. rupicola, 12 of which amplified and presented polymorphism among endemic species of Erigeron in the Archipelago. Two populations from E. rupicola and E. fernandezianus were genotyped, and one to eight alleles per locus per population were detected. The expected heterozygosity ranged from 0.000 to 0.812. •

Conclusions: These results indicate the utility of primers for cross-species populational studies in all endemic species of Erigeron in the Archipelago.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3732/ajb.1200218DOI Listing
December 2012

Progenitor-derivative speciation in Pozoa (Apiaceae, Azorelloideae) of the southern Andes.

Ann Bot 2012 Feb 23;109(2):351-63. Epub 2011 Nov 23.

Department of Systematic and Evolutionary Botany, Faculty Center of Biodiversity, University of Vienna, Austria.

Background And Aims: Studies examining patterns and processes of speciation in South America are fewer than in North America and Europe. One of the least well documented processes has been progenitor-derivative speciation. A particularly instructive example occurs in the southern Andes in the genus Pozoa (Apiaceae, Azorelloideae), which consists of only two diploid outcrossing species, the widespread P. coriacea and the geographically and ecologically restricted P. volcanica. This paper tests the hypothesis that the latter species originated from the former through local geographical and ecological isolation by progenitor-derivative speciation.

Methods: DNA sequences were analysed from Pozoa and the related South American genera Asteriscium, Eremocharis and Gymnophyton from non-coding regions of the plastid genome, ndhF-rpl32 and rpl32-trnL, plus incorporation of previously reported rpl16 intron and trnD-trnT intergenic spacer sequences. Amplified fragment length polymorphism (AFLP) data from 105 individuals in 21 populations throughout the entire range of distribution of the genus were used for estimation of genetic diversity, divergence and SplitsTree network analysis. Ecological factors, including habitat and associated species, were also examined.

Key Results: Pozoa coriacea is more similar genetically to the outgroup genera, Asteriscium and Eremocharis, than is P. volcanica. At the population level, only P. volcanica is monophyletic, whereas P. coriacea is paraphyletic. Analyses of genetic differentiation among populations and genetic divergence and diversity of the species show highest values in P. coriacea and clear reductions in P. volcanica. Pozoa coriacea occurs in several types of high elevation habitats, whereas P. volcanica is found only in newly formed open volcanic ash zones.

Conclusions: All facts support that Pozoa represents a good example of progenitor-derivative speciation in the Andes of southern South America.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/aob/mcr291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3268536PMC
February 2012

Evidence for progenitor-derivative speciation in sexually deceptive orchids.

Ann Bot 2011 Oct 2;108(5):895-906. Epub 2011 Sep 2.

Department of Systematic and Evolutionary Botany, University of Vienna, Rennweg 14, 30 Vienna, Austria.

Background And Aims: Sexually deceptive orchids of the genus Ophrys use mimicry of pollinator females to attract specific pollinators. Pollinator shifts may drive speciation in Ophrys, since novel pollinators may in principle act as isolating factors immediately. It is thus possible that evolution of novel species occurs rapidly and with a progenitor-derivative pattern. The aims of this study are to compare genetic structure and diversity among widespread and geographically restricted Ophrys taxa, to test whether genetic structure is associated with specific pollinators, and to investigate whether any widespread species may have acted as a progenitor for the evolution of more restricted taxa.

Methods: Genetic differentiation and diversity were investigated in O. leucadica and O. cinereophila, the two taxa of the Ophrys fusca sensu lato complex widespread in the Aegean, and three geographically restricted taxa from Rhodes, O. attaviria, O. parvula and O. persephonae, all differing in their specific pollinators. This was done using amplified fragment length polymorphism (AFLP) DNA fingerprinting, and sequencing of the low-copy nuclear gene LEAFY (LFY).

Key Results: All taxa were found to be separate genetic entities, with O. leucadica forming two geographic groups from the west and east of the Aegean. Genetic structure was significantly shaped by pollinators and geography, and comparison of sequence and AFLP data revealed ancestral polymorphisms shared among several taxa. Among the sampled taxa, O. leucadica harbours the greatest genetic differentiation and geographic structure, and the highest genetic diversity. Part of the genome of O. parvula, endemic to Rhodes, may be derived from O. leucadica.

Conclusions: Pollinators probably influence the genetic structure of the investigated Ophrys species. The genetic pattern identified is consistent with O. leucadica being the oldest of the sampled taxa, making O. leucadica a candidate progenitor species from which more restricted taxa such as O. parvula may have evolved.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/aob/mcr239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3177689PMC
October 2011

Phylogenetic relationships in the genus Leontopodium (Asteraceae: Gnaphalieae) based on AFLP data.

Bot J Linn Soc 2011 Apr;165(4):364-377

Institute of Pharmacy/Pharmacognosy, Faculty of Chemistry and Pharmacy, University of Innsbruck, Innrain 52c, 6020 Innsbruck, Austria.

The genus Leontopodium comprises 30-41 species. The centre of diversity is the Sino-Himalayan region in south-western China, where about 15 species occur. The two species native to Europe, L. alpinum (known as the common 'Edelweiss') and L. nivale, are part of the cultural heritage of the people living there. Despite its importance, very little is known about the systematics of the genus. Because recent molecular studies have shown that species within this genus are closely related and difficult to distinguish with rDNA and cpDNA data, we used AFLPs to obtain a more detailed understanding of the phylogeny of the genus. Our main aims were as follows: (1) to clarify species relationships within the genus; and (2) to reveal information about the biogeography of the genus. We used AFLPs with six primer combinations to investigate 216 individuals in 38 populations of 16 different species. With AFLPs, we were able to recognize 10 different groups, all of which had strong bootstrap support. These results were also congruent with the morphology-based taxonomy of the genus. Most private and rare fragments were found in the Yunnan region (south-western China) relative to Europe and Mongolia/central China, suggesting a long-lasting in situ history of populations in the centre of diversity of the genus. Our results illustrate the utility of AFLPs to resolve phylogenetic relationships between these closely related species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1095-8339.2011.01117.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3524420PMC
April 2011

Antiproliferative effects of rapamycin and celecoxib in angiosarcoma cell lines.

Anticancer Res 2010 Oct;30(10):4017-23

Department of Dermatology, University of Regensburg, Regensburg, Germany.

Background/aim: Beyond their primary field of application some well-established drugs exhibit antitumour effects in a variety of cancers. The aim of this study was to investigate the effects of the COX2 inhibitor celecoxib and the mTOR antagonist rapamycin on angiosarcoma cell lines.

Materials And Methods: Cell proliferation was measured in ASM, ISOS 1 and ISO HAS angiosarcoma cell lines with the BrdU assay.

Results: In all angiosarcoma cell lines, celecoxib as well as rapamycin inhibited cell growth in a dose-dependent manner. In ASM and ISOS 1, but not in ISO HAS angiosarcoma cells, additive growth inhibitory effects were detected by combining both agents.

Conclusion: Our results indicate that angiosarcoma cell proliferation can be inhibited by subtoxic doses of rapamycin and celecoxib. Due to their direct and stroma-mediated anticancer activities, mTOR antagonists and COX2 inhibitors represent very promising drugs in the palliative treatment of angiosarcoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
October 2010

Pleistocene refugia and polytopic replacement of diploids by tetraploids in the Patagonian and Subantarctic plant Hypochaeris incana (Asteraceae, Cichorieae).

Mol Ecol 2009 Sep 7;18(17):3668-82. Epub 2009 Aug 7.

Departamento de Biología Vegetal y Ecología, Universidad de Sevilla, 41080 Sevilla, Spain.

We report the phylogeographic pattern of the Patagonian and Subantarctic plant Hypochaeris incana endemic to southeastern South America. We applied amplified fragment length polymorphism (AFLP) and chloroplast DNA (cpDNA) analysis to 28 and 32 populations, respectively, throughout its distributional range and assessed ploidy levels using flow cytometry. While cpDNA data suggest repeated or simultaneous parallel colonization of Patagonia and Tierra del Fuego by several haplotypes and/or hybridization, AFLPs reveal three clusters corresponding to geographic regions. The central and northern Patagonian clusters (approximately 38-51 degrees S), which are closer to the outgroup, contain mainly tetraploid, isolated and highly differentiated populations with low genetic diversity. To the contrary, the southern Patagonian and Fuegian cluster (approximately 51-55 degrees S) contains mainly diploid populations with high genetic diversity and connected by high levels of gene flow. The data suggest that H. incana originated at the diploid level in central or northern Patagonia, from where it migrated south. All three areas, northern, central and southern, have similar levels of rare and private AFLP bands, suggesting that all three served as refugia for H. incana during glacial times. In southern Patagonia and Tierra del Fuego, the species seems to have expanded its populational system in postglacial times, when the climate became warmer and more humid. In central and northern Patagonia, the populations seem to have become restricted to favourable sites with increasing temperature and decreasing moisture and there was a parallel replacement of diploids by tetraploids in local populations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-294X.2009.04298.xDOI Listing
September 2009

Effective gene transfer to melanoma cells using bacterial ghosts.

Cancer Lett 2008 Apr 31;262(1):54-63. Epub 2007 Dec 31.

Cancer Research Institute, Slovak Academy of Sciences, Vlarska, Bratislava, Slovakia.

Bacterial ghosts (BG) are cell envelopes preparations of Gram-negative bacteria devoid of cytoplasmic content produced by controlled expression of PhiX174 plasmid-encoded lysis gene E. Eight melanoma cell lines were investigated for their capacity to bind and phagocyte BG derived from Escherichia coli NM522 and Mannheimia haemolytica A23. High capability to bind BG was observed in almost all of the analyzed cell lines, furthermore cells were able to take up BG independently of the used bacterial species. Further, transfection efficiency of BG loaded with DNA in vitro was measured. The Bowes cells exhibited a high expression level of GFP and the incubation of cells with plasmid loaded BG led up to 82% transfection efficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.canlet.2007.11.031DOI Listing
April 2008

Antiangiogenic and antitumor activity of a novel vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor ZD6474 in a metastatic human pancreatic tumor model.

Anticancer Drugs 2007 Jun;18(5):569-79

Department of Surgery, University of Munich-Grosshadern LMU, Munich, Germany.

ZD6474 is a novel, orally available inhibitor of vascular endothelial growth factor receptor kinase insert domain receptor/flk-1 tyrosine kinase activity with additional activity against the epidermal growth factor receptor-1 tyrosine kinase. The aim of this study was to evaluate ZD6474, alone and in combination with gemcitabine, in an orthotopic model of metastatic pancreatic cancer. Nude mice (nine to 10/group) were injected orthotopically with 1x10(6) L3.6pl human pancreatic cancer cells. Eight days later, treatment was initiated with vehicle only, gemcitabine (100 mg/kg intraperitoneal twice weekly), ZD6474 (50 mg/kg oral once daily) or a combination of the two treatments. Animals were killed on day 24 posttreatment initiation. The phosphorylation status level of vascular endothelial growth factor receptor-2 and epidermal growth factor receptor as well as the phosphorylation level of AKT and extracellular signal-regulated kinase-1/2 in different human pancreatic carcinoma cells and in human umbilical vein endothelial cells was analyzed by Western blotting. Compared with controls (1231 mg), the mean weight of treated tumors was reduced to 836, 541 and 308 mg in the gemcitabine, ZD6474 and combination groups, respectively. Lymph node metastasis was significantly reduced in both the ZD6474 alone and combined treatment groups, with 3/10 and 1/5 animals developing metastases, compared with 10/10 and 9/9 in the control and gemcitabine groups (P<0.003 and <0.0003, respectively). Microvessel density and cell proliferation were significantly reduced in the ZD6474 and combined treatment groups (P<0.02). Immunohistochemistry of tumor samples following treatment with ZD6474 resulted in a reduction of the activated and phosphorylated epidermal growth factor receptor, whereas total epidermal growth factor receptor levels were comparable with control tumors. On the basis of Western blot analysis, ZD6474 provides inhibition of tumor angiogenesis through an anti-vascular endothelial growth factor receptor-2 mechanism and inhibition of cancer cell growth through an anti-epidermal growth factor receptor mechanism. ZD6474 decreased primary pancreatic tumor growth and reduced lymph node and liver metastases compared with controls or gemcitabine alone. Tumor growth was inhibited further in animals receiving ZD6474 and gemcitabine in combination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/CAD.0b013e3280147d13DOI Listing
June 2007

Vascular targeting in pancreatic cancer: the novel tubulin-binding agent ZD6126 reveals antitumor activity in primary and metastatic tumor models.

Neoplasia 2005 Oct;7(10):957-66

Department of Surgery, Klinikum Grosshadern, Ludwig-Maximilian-University, Munich, Germany.

ZD6126 is a novel vascular-targeting agent that acts by disrupting the tubulin cytoskeleton of an immature tumor endothelium, leading to an occlusion of tumor blood vessels and a subsequent tumor necrosis. We wanted to evaluate ZD6126 in primary and metastatic tumor models of human pancreatic cancer. Nude mice were injected orthotopically with L3.6pl pancreatic cancer cells. In single and multiple dosing experiments, mice received ZD6126, gemcitabine, a combination of both agents, or no treatment. For the induction of metastatic diseases, additional groups of mice were injected with L3.6pl cells into the spleen. Twenty-four hours after a single-dose treatment, ZD6126 therapy led to an extensive central tumor necrosis, which was not seen after gemcitabine treatment. Multiple dosing of ZD6126 resulted in a significant growth inhibition of primary tumors and a marked reduction of spontaneous liver and lymph node metastases. Experimental metastatic diseases could be significantly controlled by a combination of ZD6126 and gemcitabine, as shown by a reduction of the number and size of established liver metastases. As shown by additional in vitro and in vivo experiments, possible mechanisms involve antivascular activities and subsequent antiproliferative and proapoptotic effects of ZD6126 on tumor cells, whereas direct activities against tumor cells seem unlikely. These data highlight the antitumor and antimetastatic effects of ZD6126 in human pancreatic cancer and reveal benefits of adding ZD6126 to standard gemcitabine therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1502031PMC
http://dx.doi.org/10.1593/neo.05304DOI Listing
October 2005

DNA-loaded bacterial ghosts efficiently mediate reporter gene transfer and expression in macrophages.

Mol Ther 2005 Feb;11(2):215-23

Institute of Microbiology and Genetics, Vienna University Biocenter, Dr. Bohrgasse 9, A-1030 Vienna, Austria.

There is a demand for efficient and safe DNA delivery vehicles mediating gene transfer and expression. We present bacterial ghosts as a novel platform technology for DNA delivery and targeting of macrophages. Bacterial ghosts are cell envelopes of gram-negative bacteria that are devoid of the cytoplasmic content. Escherichia coli ghosts were loaded with plasmid DNA and linear double-stranded DNA. Confocal laser scanning microscopy and flow cytometry confirmed that the DNA localized to the inner lumen of bacterial ghosts and was not associated with the outer surface of the bacteria. Up to approximately 6000 plasmids could be loaded per single ghost and the amount of loaded DNA correlated with the DNA concentration used for loading. E. coli ghosts loaded with plasmids encoding the enhanced green fluorescent protein (EGFP) targeted efficiently murine macrophages (RAW264.7) and mediated effective gene transfer. The EGFP was expressed by more than 60% of the macrophages as measured by flow cytometry detecting the green fluorescence and immunocytochemical staining with antibodies specific for EGFP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymthe.2004.09.024DOI Listing
February 2005

Bacterial ghosts as novel advanced drug delivery systems: antiproliferative activity of loaded doxorubicin in human Caco-2 cells.

J Control Release 2004 Jan;94(1):63-74

Institute for Microbiology and Genetics, Vienna Biocenter, University of Vienna, Vienna, Austria.

Systemic application of anticancer drugs often causes severe toxic side effects. To reduce the undesired effects, advanced drug delivery systems are needed which are based on specific cell targeting vehicles. In this study, bacterial ghosts from Mannheimia haemolytica were used for site-specific delivery of doxorubicin (DOX) to human colorectal adenocarcinoma cells (Caco-2). Bacterial ghosts are non-denatured envelopes of Gram-negative bacteria with fully intact surface structures for specific attachment to mammalian cells. The in vitro release profile of DOX-ghosts demonstrated that the loaded drug was non-covalently associated with the bacterial ghosts and that the drug delivery vehicles themselves represent a slow release system. Adherence studies showed that the M. haemolytica ghosts more efficiently than E. coli ghosts targeted the Caco-2 cells and released the loaded DOX within the cells. Cytotoxicity assays revealed that the DOX-ghosts exhibited potent antiproliferative activities on Caco-2 cells as the DOX associated with ghosts was two magnitude of orders more cytotoxic than free DOX provided in the medium at the same concentrations. Notably, a significant reduction in the cell viability was measured with DOX-ghosts at low DOX concentrations, which had no inhibitory effect when applied as free DOX after incubation for 16 h or when applied at higher concentrations for only 10 min to the cells. As the higher antiproliferative effects of DOX on Caco-2 cells were mediated by the specific drug targeting properties of the bacterial ghosts, the bacterial ghost system represents a novel platform for advanced drug delivery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jconrel.2003.09.010DOI Listing
January 2004

Sealed bacterial ghosts--novel targeting vehicles for advanced drug delivery of water-soluble substances.

J Drug Target 2003 Apr;11(3):151-61

Institute for Microbiology and Genetics, Vienna Biocenter, University Vienna, Austria.

The purpose of the present study was to develop a drug delivery model for water soluble drug substances using the bacterial ghost platform technology. Bacterial ghosts are non-denatured bacterial cell envelopes that are produced by the plasmid encoded gene E mediated lysis. We present a novel method to fill and seal bacterial ghosts for the application as a drug delivery system for fluid, non-anchored substances. E. coli ghosts were filled with the reporter substance calcein and sealed by fusion with membrane vesicles. By flow cytometry and fluorescence microscopy it was shown that bacterial ghosts can be filled with calcein, and that the bacterial ghosts can be sealed by restoring the membranes integrity. The adherence and uptake studies showed that almost all murine macrophages and a lower proportion of human colorectal adenocarcinoma cells took up fluorescence labeled bacterial ghosts. Moreover, these cells also took up effectively sealed E. coli ghosts filled with calcein, which then was released within the cells. Therefore, we propose bacterial ghosts as alternative drug delivery and release vehicles for advanced cell targeting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/10611860310001593366DOI Listing
April 2003

GFP-transfected tumor cells are useful in examining early metastasis in vivo, but immune reaction precludes long-term tumor development studies in immunocompetent mice.

Clin Exp Metastasis 2003 ;20(2):135-41

Department of Surgery, University of Regensburg, Germany.

To develop effective therapeutic strategies aimed at treating tumor metastasis, critical steps in this process must be better understood. For this purpose we have established a new model to visualize and quantify early metastasis. Murine CT-26 colon adenocarcinoma cells were stably transfected with green fluorescent protein (GFP). Tumor cells were intraportally delivered to the liver of Balb/c mice and subsequently tracked by intravital fluorescence microscopy. Coinjection of fluorescent beads and in vivo propidium iodide staining allowed examination of initial tumor cell arrest, extravasation, viability and proliferation. Results showed that GFP-transfection compared to conventional labeling procedures (Calcein, cytoplasmic microspheres) did not alter early metastatic properties. However, the long-term development of liver metastases expressing GFP was markedly reduced compared to wild type CT-26 tumor cells. An increase in the size and the number of liver metastases in T- and B-cell-deficient SCID mice suggested an immune response to the GFP transfected cells responsible for the reduced metastatic growth in wild-type mice. Based on our findings, this model can be used to examine the early steps of metastasis in vivo. However, in immunocompetent mice, the use of GFP-labeled tumor cells should be limited to tracking cell arrest and extravasation, whereas evaluations of long-term metastatic growth should be performed in immunodeficient mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1023/a:1022618909921DOI Listing
May 2003
-->