Immunohistochemical markers for equine granulosa cell tumors: a pilot study.

Sex cord-stromal tumors (SCSTs), generally referred to as granulosa cell tumors (GCTs) or granulosa-theca cell tumors (GTCTs) in equids, show complex compositions and variable numbers of hormone-producing cells. These tumors can be difficult to diagnose, especially in early stages. Therefore, we tested a panel of antibodies for vimentin, smooth muscle actin, laminin, Ki-67, E-cadherin, calretinin, moesin, p-ezrin, AMH, and aromatase, markers used for tumor composition and classification, progression, and prognosis in human SCSTs, on an exemplary grapefruit-size equine GCT within the left ovary of a 13-year-old mare with stallion-like behavior and elevated testosterone levels in comparison with normal ovarian tissue. The tumor showed a low proliferation rate and prominent moesin and p-ezrin staining in granulosa cells. E-cadherin, calretinin, aromatase, and AMH are suggested to be potential markers for different cell components of equine SCSTs that can support tumor diagnosis and classification.

Rapid Transfer of Plant Photosynthates to Soil Bacteria via Ectomycorrhizal Hyphae and Its Interaction With Nitrogen Availability.

Plant roots release recent photosynthates into the rhizosphere, accelerating decomposition of organic matter by saprotrophic soil microbes ("rhizosphere priming effect") which consequently increases nutrient availability for plants. However, about 90% of all higher plant species are mycorrhizal, transferring a significant fraction of their photosynthates directly to their fungal partners. Whether mycorrhizal fungi pass on plant-derived carbon (C) to bacteria in root-distant soil areas, i.e., incite a "hyphosphere priming effect," is not known. Experimental evidence for C transfer from mycorrhizal hyphae to soil bacteria is limited, especially for ectomycorrhizal systems. As ectomycorrhizal fungi possess enzymatic capabilities to degrade organic matter themselves, it remains unclear whether they cooperate with soil bacteria by providing photosynthates, or compete for available nutrients. To investigate a possible C transfer from ectomycorrhizal hyphae to soil bacteria, and its response to changing nutrient availability, we planted young beech trees (Fagus sylvatica) into "split-root" boxes, dividing their root systems into two disconnected soil compartments. Each of these compartments was separated from a litter compartment by a mesh penetrable for fungal hyphae, but not for roots. Plants were exposed to a 13C-CO2-labeled atmosphere, while 15N-labeled ammonium and amino acids were added to one side of the split-root system. We found a rapid transfer of recent photosynthates via ectomycorrhizal hyphae to bacteria in root-distant soil areas. Fungal and bacterial phospholipid fatty acid (PLFA) biomarkers were significantly enriched in hyphae-exclusive compartments 24 h after 13C-CO2-labeling. Isotope imaging with nanometer-scale secondary ion mass spectrometry (NanoSIMS) allowed for the first time in situ visualization of plant-derived C and N taken up by an extraradical fungal hypha, and in microbial cells thriving on hyphal surfaces. When N was added to the litter compartments, bacterial biomass, and the amount of incorporated 13C strongly declined. Interestingly, this effect was also observed in adjacent soil compartments where added N was only available for bacteria through hyphal transport, indicating that ectomycorrhizal fungi were acting on soil bacteria. Together, our results demonstrate that (i) ectomycorrhizal hyphae rapidly transfer plant-derived C to bacterial communities in root-distant areas, and (ii) this transfer promptly responds to changing soil nutrient conditions.

Nitrogen isotope fractionation during N uptake via arbuscular mycorrhizal and ectomycorrhizal fungi into grey alder.

Arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi affect plant nitrogen (N) dynamics. Plant N isotope patterns have been used to characterise the contribution of ECM fungi to plant N uptake. By quantifying and comparing the effects of an AM and an ECM fungus on growth, N uptake and isotopic composition of one host plant grown at different relative N supply levels, the aim of this study was to improve the mechanistic understanding of natural 15N abundance patterns in mycorrhizal plants and their underlying causes. Grey alders were inoculated with one ECM fungus or one AM fungus or left non-mycorrhizal. Plants were grown under semi-hydroponic conditions and were supplied with three rates of relative N supply ranging from deficient to luxurious. Neither mycorrhizal fungus increased plant growth or N uptake. AM root colonisation had no effect on whole plant δ15N and decreased foliar δ 15N only under N deficiency. The roots of these plants were 15N-enriched. ECM root colonisation consistently decreased foliar and whole plant δ15N. It is concluded, that both mycorrhizal fungi contributed to plant N uptake into the shoot. Nitrogen isotope fractionation during N assimilation and transformations in fungal mycelia is suggested to have resulted in plants receiving 15N-depleted N via the mycorrhizal uptake pathways. Negative mycorrhizal growth effects are explained by symbiotic resource trade on carbon and N and decreased direct plant N uptake.

The relationship between N isotopic fractionation within soybean and N2 fixation during soybean development.

The contribution of N(2) fixation to overall soybean N uptake has most commonly been quantified by N isotope-based methods, which rely on isotopic differences in plant N between legumes and non-fixing reference plants. The choice of non-fixing reference plants is critical for the accuracy of isotope-based methods, and mismatched reference plants remain a potential source of error. Accurate estimates of soybean N(2) fixation also require information on N isotopic fractionation within soybean. On the basis of a previous observation of a close correlation between an expression of N fractionation within soybean and the proportion of plant N derived from atmosphere (%Ndfa) determined by (15) N natural abundance, this field study aimed at assessing the relationship between various expressions describing intraplant (15) N or N partitioning and %Ndfa during soybean development. Starting from a late vegetative stage until beginning senescence, the N content and N isotopic composition of shoots, roots and nodules of nodulated and non-nodulated soybeans was determined at eight different developmental stages. Regression analysis showed that %Ndfa most closely correlated with the difference in the N isotopic composition of shoot N minus that of root including nodule N, and that this relationship was similar to that obtained in a previous multi-site field study. We therefore consider this expression to hold promise as a means of quantifying %Ndfa independent of a reference plant, which would avoid some of the external sources of error introduced by the use of reference plants in determining %Ndfa.

Common genomic aberrations in basaloid squamous cell carcinoma and carcinosarcoma of the esophagus detected by CGH and array CGH.

Basaloid squamous cell carcinoma (BSCC) and carcinosarcoma of the esophagus are rare entities, making up fewer than 2% of esophageal malignancies. Comparative genomic hybridization (CGH) in 1 case of BSCC and 2 cases of carcinosarcoma and subsequent array CGH in 1 case each of BSCC and carcinosarcoma revealed common chromosomal gains at 2p25.3-2p12, 7q21.3-7q22.3, and 11q13.2-11q13.4. Chromosomal losses at 13q31qter were observed in both carcinosarcomas. In addition, progression of genomic instability from in situ to invasive carcinosarcoma could be demonstrated by using array CGH. Our observations suggest a common genetic origin of BSCC and carcinosarcoma.

Belowground carbon allocation by trees drives seasonal patterns of extracellular enzyme activities by altering microbial community composition in a beech forest soil.

*Plant seasonal cycles alter carbon (C) and nitrogen (N) availability for soil microbes, which may affect microbial community composition and thus feed back on microbial decomposition of soil organic material and plant N availability. The temporal dynamics of these plant-soil interactions are, however, unclear. *Here, we experimentally manipulated the C and N availability in a beech forest through N fertilization or tree girdling and conducted a detailed analysis of the seasonal pattern of microbial community composition and decomposition processes over 2 yr. *We found a strong relationship between microbial community composition and enzyme activities over the seasonal course. Phenoloxidase and peroxidase activities were highest during late summer, whereas cellulase and protease peaked in late autumn. Girdling, and thus loss of mycorrhiza, resulted in an increase in soil organic matter-degrading enzymes and a decrease in cellulase and protease activity. *Temporal changes in enzyme activities suggest a switch of the main substrate for decomposition between summer (soil organic matter) and autumn (plant litter). Our results indicate that ectomycorrhizal fungi are possibly involved in autumn cellulase and protease activity. Our study shows that, through belowground C allocation, trees significantly alter soil microbial communities, which may affect seasonal patterns of decomposition processes.

Zinc accumulation potential and toxicity threshold determined for a metal-accumulating Populus canescens clone in a dose-response study.

The effect of increasing soil Zn concentrations on growth and Zn tissue concentrations of a metal-accumulating aspen clone was examined in a dose-response study. Plants were grown in a soil with a low native Zn content which was spiked with Zn salt solutions and subsequently aged. Plant growth was not affected by NH(4)NO(3)-extractable soil Zn concentrations up to 60 microg Zn g(-1) soil, but it was completely inhibited at extractable concentrations above 90 microg Zn g(-1) soil. From these data an effective concentration of 68.5 microg extractable Zn g(-1) soil was calculated at which plant growth was reduced by 50%. The obtained information on toxicity threshold concentrations, and the relation between plant Zn accumulation and extractable soil Zn concentrations may be used to assess the suitability of the investigated Populus canescens clone for various phytoremediation strategies. The potential risk of metal transfer into food webs associated with P. canescens stands on Zn-polluted sites may also be estimated.

Bioconcentration of zinc and cadmium in ectomycorrhizal fungi and associated aspen trees as affected by level of pollution.

Concentrations of Zn and Cd were measured in fruitbodies of ectomycorrhizal (ECM) fungi and leaves of co-occurring accumulator aspen. Samples were taken on three metal-polluted sites and one control site. Fungal bioconcentration factors (BCF = fruitbody concentration: soil concentration) were calculated on the basis of total metal concentrations in surface soil horizons (BCF(tot)) and NH(4)NO(3)-extractable metal concentrations in mineral soil (BCF(lab)). When plotted on log-log scale, values of BCF decreased linearly with increasing soil metal concentrations. BCF(lab) for both Zn and Cd described the data more closely than BCF(tot). Fungal genera differed in ZnBCF but not in CdBCF. The information on differences between fungi with respect to their predominant occurrence in different soil horizons did not improve relations of BCF with soil metal concentrations. Aspen trees accumulated Zn and Cd to similar concentrations as the ECM fungi. Apparently, the fungi did not act as an effective barrier against aspen metal uptake by retaining the metals.

Ectomycorrhizal communities associated with Populus tremula growing on a heavy metal contaminated site.

European aspen is one of the most widely distributed trees in Central Europe and is a typical early colonizer of poor and disturbed soils. However, little is known about ectomycorrhizal (ECM) fungi in these ecosystems. We examined the ECM community of European aspen growing on a heavily contaminated site in southern Austria by analysing ECM roots, sorting them into morphotypes, subjecting them to DNA extraction, PCR, and DNA sequencing. ECM root symbionts were sampled two times in 2004. During this time, the below-ground community structure was relatively stable; we found no evidence of taxa adapted to summer or autumn conditions and only two species varied widely in occurrence between soil horizons. The ECM fungal community was diverse (54 species), rich in Basidiomycota (43 species), and dominated by Cenococcum geophilum and fungi with corticoid basidiomes (e.g. Thelephoraceae).

Media formulation influences in vitro ectomycorrhizal synthesis on the European aspen Populus tremula L.

The effect of various media formulations on in vitro ectomycorrhizal synthesis of identified fungal strains with European aspen (Populus tremula L.) was tested in Petri dishes. Pre-grown seedlings were transferred to various nutrient media and inoculated with Paxillus involutus isolates using modified sandwich techniques. Mycorrhiza formation was evaluated macroscopically and further confirmed by microscopic examination of semi-thin sections for anatomical features of the mantle and the Hartig net. Standard media formulations did not support successful ectomycorrhiza formation because of either very poor plant survival (below 20%) or impaired fungal growth. The inclusion of micronutrients and vitamins in a Melin Norkrans (MMN)-based medium increased plant survival rate to above 60% and supported successful mycorrhizal synthesis. P. involutus isolates formed mycorrhizas with a characteristic Hartig net restricted to the epidermis. Mantle density and thickness varied depending on the isolate. In a follow-up experiment, the adapted medium supported successful ectomycorrhiza formation by various Laccaria and Hebeloma isolates. Our results show that an exogenous supply of vitamins and micronutrients in the medium was a prerequisite for successful mycorrhization of P. tremula in vitro in Petri dishes.

Flavonoids and strigolactones in root exudates as signals in symbiotic and pathogenic plant-fungus interactions.

Secondary plant compounds are important signals in several symbiotic and pathogenic plant-microbe interactions. The present review is limited to two groups of secondary plant compounds, flavonoids and strigolactones, which have been reported in root exudates. Data on flavonoids as signaling compounds are available from several symbiotic and pathogenic plant-microbe interactions, whereas only recently initial data on the role of strigolactones as plant signals in the arbuscular mycorrhizal symbiosis have been reported. Data from other plant-microbe interactions and strigolactones are not available yet. In the present article we are focusing on flavonoids in plant-fungal interactions such as the arbuscular mycorrhizal (AM) association and the signaling between different Fusarium species and plants. Moreover the role of strigolactones in the AM association is discussed and new data on the effect of strigolactones on fungi, apart from arbuscular mycorrhizal fungi (AMF), are provided.

Colonisation and molecular diversity of arbuscular mycorrhizal fungi in the aquatic plants Littorella uniflora and Lobelia dortmanna in southern Sweden.

The colonisation intensity and composition of the mycorrhizal community in the aquatic plants Lobelia dortmanna and Littorella uniflora were studied. The mycorrhizal fungi were characterised by fungal specific nested PCR and sequencing using the 5'-end of the LSU rDNA as target. For this, primers for the clade of Acaulospora, the clade including Glomus mosseae and G. intraradices and the clade containing G. etunicatum and G. claroideum were used. The nested PCR products were screened for different sequence types using single stranded conformation polymorphism (SSCP) and representatives for each type were sequenced. A phylogenetic analysis of the sequences showed two phylotypes of Acaulospora, one phylotype within the clade of G. etunicatum/G. claroideum and five within the G. mosseae/ G. intraradices clade. The colonisation intensity was comparable to that seen in typical grassland vegetation. The neutral lipid fatty acid 16: 1omega5 was seen to be indicative of mycorrhizal colonisation with concentrations up to 35 nmol mg(-1) root DW, which indicates that the fungi are active.

Rhizosphere characteristics of the arsenic hyperaccumulator Pteris vittata L. and monitoring of phytoremoval efficiency.

Recently discovered As-hyperaccumulator ferns hold promise for phytoremediation of As-polluted soils. We investigated changes in the rhizosphere characteristics of Pteris vittata (Chinese Brake fern) relevant for its use in phytoextraction. Plants were grown in rhizoboxes filled with soil containing 2270 mg kg(-1) As. Dissolved organic carbon (DOC) concentrations in rhizosphere soil solution were increased by 86% and appeared to enhance total Fe solubility due to complexation reactions. Despite substantial removal of As by the fern, As was not significantly decreased in the rhizsophere soil solution after one cropping, apparently due to the large buffer capacity of the soil and possibly because of ion competition with DOC. However, the difference between 0.05 M (NH4)2SO4-extractable labile As in bulk and rhizosphere soil accounted for 8.9% of total As accumulated in the fern, indicating that As was mainly acquired from less available pools. Moreover, As depletion in the rhizosphere and limited resupply from less available pools were indicated by a 19.3% decreased As flux, measured using the technique of diffusive gradients in thin films (DGT). Modeling of the DGT-soil system was able to show that the rate of release from solid phase to solution in the rhizosphere was one-third of that in the bulk soil. Applying the remedial strategy of bioavailable contaminant stripping, which aims at diminishing the phytoavailable pollutant fraction, DGT can be used as a monitoring tool to evaluate the efficiency of phytoextraction and to study the potential resupply of bioavailable pools after phytoextraction has ceased.

Laryngotracheoscopic findings in long-term follow-up after Griggs tracheostomy.

OBJECTIVE: Analysis of laryngotracheoscopic findings of the upper airway tract following percutaneous tracheostomy using the technique according to Griggs. DESIGN: Retrospective cohort study PATIENTS: Nineteen of 32 long-term surviving patients (mean follow-up duration, 17 months; range, 11 to 23 months) underwent a modified Griggs tracheostomy during their stay in the ICU following cardiothoracic surgery. INTERVENTIONS: Nineteen patients gave their informed consent for laryngotracheoscopy to localize and assess the percutaneous dilatational tracheostomy (PDT) puncture site, to evaluate the laryngotracheal morphology, and to quantify tracheal stenosis if present. In addition, specific symptoms of the upper airway tract were evaluated. RESULTS: At the time of examination, no clinically relevant cases of stenoses were found, although one patient had undergone surgical revision of the PDT for extensive granulation prior to our examination. The endoscopic examination revealed that 12 of 19 patients (63%) had tracheal stenoses > 10%, and 2 patients had tracheal stenoses > 25%. In 7 of 19 patients (32%), the cricoid cartilage was affected by the PDT site. Despite endoscopic guidance during PDT, the location of the puncture site was found to vary greatly. CONCLUSION: In contrast to recent reports on the long-term outcome after Griggs PDT, we found tracheal stenoses > 10% in 63% of our patients. The grade of stenosis depended mainly on the puncture site of the PDT. Based on these results, we would emphasize the importance of adequate endoscopic guidance during PDT. Further studies are required in order to clarify the risk of long-term complications arising after PDT using the technique of Griggs.

Long-term outcome after Griggs tracheostomy.

OBJECTIVE: The purpose of this study was to evaluate laryngotracheal stenoses in the long-term outcome after percutaneous tracheostomy. METHODS: Between 1997 and 2000, 162 patients were tracheostomized during their postoperative stay at the intensive care unit of the Department of Cardiac Surgery, University of Heidelberg. Thirty-eight of 80 long-term surviving patients (mean follow-up: 22 months, range: 7-50 months) gave their informed consent to follow-up laryngotracheoscopy. By using this technique, we localized the tracheostomy site, evaluated the laryngotracheal morphology, and quantified laryngotracheal stenosis planimetrically. RESULTS: Clinically relevant stenoses were found in one patient. Another patient had undergone surgical revision of the percutaneous dilatational tracheostomy (PDT) prior to our examination. The endoscopic examination revealed that 89.5% (34/38) of the patients exhibited tracheal stenosis, less than 25% without clinical symptoms. Despite endoscopic guidance during PDT, the location of the puncture site was found to vary greatly. Cricoidal lesions were identified in 15 patients. In only 12 patients (31.6%), the PDT had been placed at the optimal location between the first and the second tracheal ring. In these patients, we found the lowest rate of tracheal stenosis in tracheotomies without fractured tracheal rings. CONCLUSION: Since clinically relevant tracheal stenosis has been found to depend mainly on the puncture site of the PDT and tracheal fractures during PDT, we want to emphasize the importance of adequate endoscopic guidance during and the careful performance of the PDT. Further follow-up studies are necessary to improve and ensure the quality of PDT techniques.