A comparison of soil amendment with either anaerobically digested or fresh cattle manure and its impact on genetic diversity, microbial activity and physiological community profile

Póster presentado en el congreso I Global Soil Biodiversity Conference, celebrado en Dijon, Francia, del 2 al 5 de diciembre de 2014In recent years, small-and mid-scale biogas plants have thrived in Europe and led to a change in land-use. Manures that used to be applied to agricultural soils are now used for energy generation in biogas reactors and instead digestateis applied to agricultural soils. Here we present the results of a study simulating soil amendment with either anaerobically digested or fresh cattle manure and its effect on the microbial community.Peer Reviewe

Editorial for special issue "Unleashing the Hidden Potential of Anaerobic Fungi"

Anaerobic fungi (AF) of the phylum Neocallimastigomycota are a very peculiar group of microorganisms. Since their first discovery in the early nineteen-hundreds and assignment to the kingdom Fungi in 1975 by Orpin [1], many researchers have delved into these highly potent degraders of lignocellulosic biomass (LCB). Their panoply of hydrolytic enzymes makes them key players in the digestive tract of herbivores, but their occurrence may not be restricted to this habitat alone. Despite the plenitude of research on this AF group, many questions still remain unanswered, and the implementation of AF within, e.g., biomethanation of LCB or bioethanol production, is still in its infancy. This is where international projects such as “Unleashing the Hidden Potential of Anaerobic Fungi” (https://www.hipoaf.com; accessed last on 14 February 2023) hook in and aim at answering basic questions such as ideal growth conditions, improved and novel detection techniques, screening for novel habitats, strains and enzymes, symbiotic interactions of AF, and, eventually, at paving the way to the successful biotechnological implementation of these unique microorganisms. These questions are also the scope of this Special Issue, comprising eight original articles and two reviews that are dedicated to recent updates on various fields of AF research. The contributions span from AF in animal husbandry and biotechnology over AF systematics, physiology and molecular detection to isolation strategies

The use of extracellular DNA as a proxy for specific microbial activity

The ubiquity and relevance of extracellular DNA (exDNA) are well-known and increasingly gaining importance in many fields of application such as medicine and environmental microbiology. Although sources and types of exDNA are manifold, ratios of specific DNA-molecules inside and outside of living cells can give reliable information about the activity of entire systems and of specific microbial groups or species. Here, we introduce a method to discriminate between internal (iDNA), as well as bound and free exDNA, and evaluate various DNA fractions and related ratios (ex:iDNA) regarding their applicability to be used as a fast, convenient, and reliable alternative to more tedious RNA-based activity measurements. In order to deal with microbial consortia that can be regulated regarding their activity, we tested and evaluated the proposed method in comparison to sophisticated dehydrogenase- and RNA-based activity measurements with two anaerobic microbial consortia (anaerobic fungi and syntrophic archaea and a microbial rumen consortium) and three levels of resolution (overall activity, total bacteria, methanogenic archaea). Furthermore, we introduce a 28S rRNA gene-specific primer set and qPCR protocol, targeting anaerobic fungi (Neocallimastigomycota). Our findings show that the amount of actively released free exDNA (fDNA) strongly correlates with different activity measurements and is thus suggested to serve as a proxy for microbial activity.publishersversionPeer reviewe

The effect of a high-grain diet on the rumen microbiome of goats with a special focus on anaerobic fungi

This work investigated the changes of the rumen microbiome of goats switched from a forage to a concentrate diet with special attention to anaerobic fungi (AF). Female goats were fed an alfalfa hay (AH) diet (0% grain; n = 4) for 20 days and were then abruptly shifted to a high-grain (HG) diet (40% corn grain, 60% AH; n = 4) and treated for another 10 days. Rumen content samples were collected from the cannulated animals at the end of each diet period (day 20 and 30). The microbiome structure was studied using high-throughput sequencing for bacteria, archaea (16S rRNA gene) and fungi (ITS2), accompanied by qPCR for each group. To further elucidate unclassified AF, clone library analyses were performed on the ITS1 spacer region. Rumen pH was significantly lower in HG diet fed goats, but did not induce subacute ruminal acidosis. HG diet altered prokaryotic communities, with a significant increase of Bacteroidetes and a decrease of Firmicutes. On the genus level Prevotella 1 was significantly boosted. Methanobrevibacter and Methanosphaera were the most abundant archaea regardless of the diet and HG induced a significant augmentation of unclassified Thermoplasmatales. For anaerobic fungi, HG triggered a considerable rise in Feramyces observed with both ITS markers, while a decline of Tahromyces was detected by ITS2 and decrease of Joblinomyces by ITS1 only. The uncultured BlackRhino group revealed by ITS1 and further elucidated in one sample by LSU analysis, formed a considerable part of the AF community of goats fed both diets. Results strongly indicate that the rumen ecosystem still acts as a source for novel microorganisms and unexplored microbial interactions and that initial rumen microbiota of the host animal considerably influences the reaction pattern upon diet change.Fil: Fliegerova, Katerina O.. Czech Academy of Sciences; República ChecaFil: Podmirseg, Sabine M.. Universidad de Innsbruck; AustriaFil: Vinzelj, Julia. Universidad de Innsbruck; AustriaFil: Grilli, Diego Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaFil: Kvasnová, Simona. Czech Academy of Sciences; República ChecaFil: Schierová, Dagmar. Czech Academy of Sciences; República ChecaFil: Sechovcová, Hana. Czech Academy of Sciences; República ChecaFil: Mrázek, Jakub. Czech Academy of Sciences; República ChecaFil: Siddi, Giuliana. Università degli Studi di Sassari; ItaliaFil: Arenas, Graciela Nora. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaFil: Moniello, Giuseppe. Università degli Studi di Sassari; Itali

FLT3 and FLT3-ITD phosphorylate and inactivate the cyclin-dependent kinase inhibitor p27Kip1 in acute myeloid leukemia

P27Kip1 (p27) can prevent cell proliferation by inactivating cyclin-dependent kinases. This function is impaired upon phosphorylation of p27 at tyrosine residue 88. We observed that FLT3 and FLT3-ITD can directly bind and selectively phosphorylate p27 on this residue. Inhibition of FLT3-ITD in cell lines strongly reduced p27 tyrosine 88 phosphorylation and resulted in increased p27 levels and cell cycle arrest. Subsequent analysis revealed the presence of tyrosine 88 phosphorylated p27 in primary patient samples. Inhibition of FLT3 kinase activity with AC220 significantly reduced p27 tyrosine 88 phosphorylation in cells isolated from FLT3 wild type expressing acute myeloid leukemia (AML) patients. In FLT3-ITD positive AML patients, p27 tyrosine 88 phosphorylation was reduced in 5 out of 9 subjects, but, surprisingly, was increased in 4 patients. This indicated that other tyrosine kinases such as Src family kinases might contribute to p27 tyrosine 88 phosphorylation in FLT3-ITD positive AML cells. In fact, incubation with the Src family kinase inhibitor dasatinib could decrease p27 tyrosine 88 phosphorylation in these patient samples, indicating that p27 phosphorylated on tyrosine 88 may be a therapeutic marker for the treatment of AML patients with tyrosine kinase inhibitors

No time to die : comparative study on preservation protocols for anaerobic fungi

Anaerobic fungi (AF, phylum Neocallimastigomycota) are best known for their ability to anaerobically degrade recalcitrant lignocellulosic biomass through mechanic and enzymatic means. While their biotechnological potential is well-recognized, applied research on AF is still hampered by the time-consuming and cost-intensive laboratory routines required to isolate, maintain, and preserve AF cultures. Reliable long-term preservation of specific AF strains would aid basic as well as applied research, but commonly used laboratory protocols for AF preservation can show erratic survival rates and usually exhibit only moderate resuscitation success for up to one or two years after preservation. To address both, the variability, and the preservation issues, we have set up a cross-laboratory, year-long study. We tested five different protocols for the preservation of AF. The experiments were performed at three different laboratories (Austria, Germany, Switzerland) with the same three morphologically distinct AF isolates (Anaeromyces mucronatus, Caeocmyces sp., and Neocallimastix cameroonii) living in stable co-culture with their naturally occurring, syntrophic methanogens. We could show that handling greatly contributes to the variability of results, especially in Anaeromyces mucronatus. Cryopreservation of (mature) biomass in liquid nitrogen had the highest overall survival rates (85–100%, depending on the strain and laboratory). Additionally, preservation on agar at 39°C had surprisingly high survival rates for up to 9 months, if pieces of agar containing mature AF thalli were resuscitated. This low-cost, low-effort method could replace consecutive batch cultivation for periods of up to 6 months, while long-term preservation is best done by cryopreservation in liquid nitrogen. Regardless of the method, however, preserving several replicates (>three) of the same strain is highly advisable

CoMA - an intuitive and user-friendly pipeline for amplicon-sequencing data analysis

In recent years, there has been a veritable boost in next-generation sequencing (NGS) of gene amplicons in biological and medical studies. Huge amounts of data are produced and need to be analyzed adequately. Various online and offline analysis tools are available; however, most of them require extensive expertise in computer science or bioinformatics, and often a Linux-based operating system. Here, we introduce “CoMA–Comparative Microbiome Analysis” as a free and intuitive analysis pipeline for amplicon-sequencing data, compatible with any common operating system. Moreover, the tool offers various useful services including data pre-processing, quality checking, clustering to operational taxonomic units (OTUs), taxonomic assignment, data post-processing, data visualization, and statistical appraisal. The workflow results in highly esthetic and publication-ready graphics, as well as output files in standardized formats (e.g. tab-delimited OTU-table, BIOM, NEWICK tree) that can be used for more sophisticated analyses. The CoMA output was validated by a benchmark test, using three mock communities with different sample characteristics (primer set, amplicon length, diversity). The performance was compared with that of Mothur, QIIME and QIIME2-DADA2, popular packages for NGS data analysis. Furthermore, the functionality of CoMA is demonstrated on a practical example, investigating microbial communities from three different soils (grassland, forest, swamp). All tools performed well in the benchmark test and were able to reveal the majority of all genera in the mock communities. Also for the soil samples, the results of CoMA were congruent to those of the other pipelines, in particular when looking at the key microbial players.Ministerio de Economía, Industria y Competitividad | Ref. RYC-2016-2123