Assessment of soil bacterial communities with emphasis on the phylum Acidobacteria

The seasonal culturability (February, April, August) of bacterial cells from a microbial community of an alpine calcareous soil was assessed employing the MicroDrop technique using different laboratory media with humic acid analogs (HA), a mixture of polymers (POL), artificial root exudates (RO), nutrient broth, or soil extract as carbon and energy sources. Thereby, the summer August sample showed the highest culturability value in media supplemented with soil extract (13.5%). Since only 81 wells of a total number of 1008 individual growth tests were overgrown with the February soil sample, the cultivation success was the lowest for the winter environment (0.16%). The major aim of the present study, however, was to assess the cultivation success for cells even exposed to extreme environmental conditions by using defined media. Therefore, subsequent analysis focused on the cultures obtained from the February sample and in media supplemented with RO. It was shown that the monomeric organic carbon of RO proved to be superior to POL and HA for the optimization of the cultivation success (i.e., 71 of the total number of 81 cultures). The quantitative PCR approach confirmed the high coverage of the present analysis since the target groups (Firmicutes, Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Acidobacteria) constituted 73.6% of all eubacteria in the sample whereas the major part was composed of Alphaproteobacteria (49.2%) and Acidobacteria (20.1%). A total of 251 bacteria were analyzed representing 53 distinct phylotypes of which 73% are previously unknown. The majority of the cultured fraction was closely related to the Alphaproteobacteria with the largest number of different phylotypes and the highest evenness value. Although this phylum dominated the cultivated fraction, its cultivation success was hundredfold lower than its abundance in the natural community (0.4% of total cell numbers). Also the Bacteroidetes were most frequently cultured but were dominated by one phylotype (Sphingoterrabacterium pocheensis). The relative culturability of the Bacteroidetes was the highest of all groups and reached 25% of the numbers detected by real-time PCR. The lowest culturability was assessed for the Acidobacteria with only one single cultivated phylotype using media with POL supplemented with signal compounds. However, this phylotype represents a novel, previously unknown acidobacterium, strain Jbg-1. The phylum Acidobacteria mostly consists of environmental 16S rRNA gene sequences and so far comprises only the four validly described species Holophaga foetida, Geothrix fermentans, Acidobacterium capsulatum and Terriglobus roseus. In the present thesis two different novel strains of acidobacteria were isolated. Strain Jbg-1 and the second strain Wbg-1, which was recovered from a coculture with a methanotrophic bacterium established from calcareous forest soil. Both strains represent members of subdivision 1 of the phylum Acidobacteria and are closely related to each other (98.0 % 16S rRNA gene sequence similarity). At a sequence similarity of 93.8-94.7%, strains Jbg-1 and Wbg-1 are only distantly related to the closest described relative, Terriglobus roseus, and accordingly are described as members of the novel genus Edaphobacter gen. nov. Based on the DNA-DNA-similarity between strains Jbg-1 and Wbg-1 of 11.5-13.6% and their chemotaxonomic and phenotypic characteristics, the two strains are assigned to two separate species, Edaphobacter modestus sp. nov. with strain Jbg-1T (= ATCC BAA-1329T = DSM 18101T) as the type strain, and E. aggregans sp. nov. with strain Wbg-1T (= ATCC BAA-1497T = DSM 19364T) as the type strain. The two novel species are adapted to low carbon concentrations and to neutral to slightly acidic conditions. It was shown that strain Jbg-1 was also well adapted to long-term survival and to higher carbon concentrations after subcultivation. Unexpectedly, a high percentage of interspecific interaction was obtained for the cultivation approach of the February alpine soil (75% cocultures), which represented the major reason for the low cultivation success. Only 16 out of 71 cultures with RO consisted of single cultivated strains. Due to the frequent occurrence of different bacteria in the same cultures, the actual cultivation success was 4.9 fold higher than the value calculated from the abundance of positive cultures. For subsequent analysis, the effect of different treatments during the cultivation approach on the number and composition of bacteria cultured was investigated. In order to differentiate between free-living and attached cells, bacteria were detached from soil particles and used to set up parallel incubations. The detachment from soil particles prior to inoculation had no effect on the total cultivation success and on co-cultivation. Furthermore, signal compounds (cyclic AMP and N-butyryl homoserine lactone), however, increased the cultivation success and co-culturability. Addition of signal compounds yielded different types of activated bacteria and enhanced the total number of phylotypes per co-culture towards 4, 5, 6, and 7 different bacteria. The major part of the single cultivated strains represented a single phylotype, which was related to Sphingoterrabacterium pocheensis. In contrast, most co-cultures contained members of the Alpha- and Betaproteobacteria whereas relatives of Phyllobacterium brassicacearum, Rhodospirillum rubrum, Inqulinus ginsengisoli, Delftia tsuruhatensis, and Rhodocyclus tenuis were the most abundant ones. In conclusion, it is supposed that cell-to-cell interaction routinely occurs between different species of microorganisms, although the way, how these aerobic microorganisms beneficially interact remained to be shown. The elucidation of such interactions seems to be the most successful approach to enhance the culturability of interesting soil bacteria to promote their growth in pure or defined co-cultures

TLR3 and TLR4 expression in healthy and diseased human endometrium

<p>Abstract</p> <p>Background</p> <p>Toll-like receptors (TLRs) play an essential role in the innate immune system by initiating and directing immune response to pathogens. TLRs are expressed in the human endometrium and their regulation might be crucial for the pathogenesis of endometrial diseases.</p> <p>Methods</p> <p>TLR3 and TLR4 expression was investigated during the menstrual cycle and in postmenopausal endometrium considering peritoneal endometriosis, hyperplasia, and endometrial adenocarcinoma specimens (grade 1 to 3). The expression studies applied quantitative RT-PCR and immunolabelling of both proteins.</p> <p>Results</p> <p>TLR3 and TLR4 proteins were mostly localised to the glandular and luminal epithelium. In addition, TLR4 was present on endometrial dendritic cells, monocytes and macrophages. TLR3 and TLR4 mRNA levels did not show significant changes during the menstrual cycle. In patients with peritoneal endometriosis, TLR3 and TLR4 mRNA expression decreased significantly in proliferative diseased endometrium compared to controls. Interestingly, ectopic endometriotic lesions showed a significant increase of TLR3 und TLR4 mRNA expression compared to corresponding eutopic tissues, indicating a local gain of TLR expression. Endometrial hyperplasia and adenocarcinoma revealed significantly reduced receptor levels when compared with postmenopausal controls. The lowest TLR expression levels were determined in poor differentiated carcinoma (grade 3).</p> <p>Conclusion</p> <p>Our data suggest an involvement of TLR3 and TLR4 in endometrial diseases as demonstrated by altered expression levels in endometriosis and endometrial cancer.</p

Distribution of SiO2 nanoparticles in 3D liver microtissues

Introduction: Nanoparticles (NPs) are used in numerous products in technical fields and biomedicine; their potential adverse effects have to be considered in order to achieve safe applications. Besides their distribution in tissues, organs, and cellular localization, their impact and penetration during the process of tissue formation occurring in vivo during liver regeneration are critical steps for establishment of safe nanomaterials. Materials and methods: In this study, 3D cell culture of human hepatocarcinoma cells (HepG2) was used to generate cellular spheroids, serving as in vitro liver microtissues. In order to determine their differential distribution and penetration depth in HepG2 spheroids, SiO2 NPs were applied either during or after spheroid formation. The NP penetration was comprehensively studied using confocal laser scanning microscopy and scanning electron microscopy. Results: Spheroids were exposed to 100 µg mL-1 SiO2 NPs either at the beginning of spheroid formation, or during or after formation of spheroids. Microscopy analyses revealed that NP penetration into the spheroid is limited. During and after spheroid formation, SiO2 NPs penetrated about 20 µm into the spheroids, corresponding to about three cell layers. In contrast, because of the addition of SiO2 NPs simultaneously to cell seeding, NP agglomerates were located also in the spheroid center. Application of SiO2 NPs during the process of spheroid formation had no impact on final spheroid size. Conclusion: Understanding the distribution of NPs in tissues is essential for biomedical applications. The obtained results indicate that NPs show only limited penetration into already formed tissue, which is probably caused by the alteration of the tissue structure and cell packing density during the process of spheroid formation

Light-Powered Reactivation of Flagella and Contraction of Microtubule Networks: Toward Building an Artificial Cell

Artificial systems capable of self-sustained movement with self-sufficient energy are of high interest with respect to the development of many challenging applications, including medical treatments, but also technical applications. The bottom-up assembly of such systems in the context of synthetic biology is still a challenging task. In this work, we demonstrate the biocompatibility and efficiency of an artificial light-driven energy module and a motility functional unit by integrating light-switchable photosynthetic vesicles with demembranated flagella. The flagellar propulsion is coupled to the beating frequency, and dynamic ATP synthesis in response to illumination allows us to control beating frequency of flagella in a light-dependent manner. In addition, we verified the functionality of light-powered synthetic vesicles in in vitro motility assays by encapsulating microtubules assembled with force-generating kinesin-1 motors and the energy module to investigate the dynamics of a contractile filamentous network in cell-like compartments by optical stimulation. Integration of this photosynthetic system with various biological building blocks such as cytoskeletal filaments and molecular motors may contribute to the bottom-up synthesis of artificial cells that are able to undergo motor-driven morphological deformations and exhibit directional motion in a light-controllable fashion.R.A., V.N., E.B., I.G., and A.G. acknowledge support from the European Union’s Horizon 2020 research and innovation programme under grant agreement MAMI No. 766007. C.K., A.B., E.B., K.S., I.G., T.V.K., and A.G. thank MaxSynBio Consortium, which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Societ

Pathogenicity of Pythium species to maize

AbstractPythium isolates from diseased and dead bait plants of maize and cress grown in compost or various soils (maize fields, parkland under deciduous trees, grassland) were characterised and tested for pathogenicity to maize (Zea mays L.). In pot tests performed under controlled conditions, pathogenicity of the isolates to maize was apparent by reduction of root and shoot growth, whereas damping-off of maize seedlings was less frequent. Contrarily, pea seedlings were killed by pathogenic Pythium isolates. Pythium isolates from diseased maize seedlings and pathogenic strains from other gramineous plants (P. phragmitis, P. aff.phragmitis, P. catenulatum) were not necessarily more virulent to maize compared to isolates originating from dicotyledonous plants (cress). The most virulent isolates originated from compost and caused a reduction of maize shoot growth of up to 60%. Phylogenetic analysis revealed that they were very closely related to P. ultimum var. ultimum and P. arrhenomanes, respectively. Isolates originating from maize fields, grassland and parkland under deciduous trees, a reference culture of P. arrhenomanes and strains of P. phragmitis, P. aff. phragmitis and P. catenulatum with known pathogenicity on reed were non-pathogenic on maize. Isolates from compost, and from maize fields generally had a higher temperature optimum for mycelial growth (30 °C) and a faster growth rate (1.5–2.0 mm h−1) compared to the isolates from parkland under deciduous trees and grassland soil (20–25 °C, ~1.0 mm h−1), respectively. This study indicates a potential impact of pathogenic Pythium on maize plants even in the absence of visible symptoms

Targeted T1 Magnetic Resonance Imaging Contrast Enhancement with Extraordinarily Small CoFe2O4 Nanoparticles

Extraordinarily small (2.4 nm) cobalt ferrite nanoparticles (ESCIoNs) were synthesized by a one-pot thermal decomposition approach to study their potential as magnetic resonance imaging (MRI) contrast agents. Fine size control was achieved using oleylamine alone, and annular dark-field scanning transmission electron microscopy revealed highly crystalline cubic spinel particles with atomic resolution. Ligand exchange with dimercaptosuccinic acid rendered the particles stable in physiological conditions with a hydrodynamic diameter of 12 nm. The particles displayed superparamagnetic properties and a low r2/r1 ratio suitable for a T1 contrast agent. The particles were functionalized with bile acid, which improved biocompatibility by significant reduction of reactive oxygen species generation and is a first step toward liver-targeted T1 MRI. Our study demonstrates the potential of ESCIoNs as T1 MRI contrast agents

Towards a standardization of biomethane potential tests

Production of biogas from different organic materials is a most interesting source of renewable energy. The biomethane potential (BMP) of these materials has to be determined to get insight in design parameters for anaerobic digesters. Although several norms and guidelines for BMP tests exist, inter-laboratory tests regularly show high variability of BMPs for the same substrate. A workshop was held in June 2015, in Leysin, Switzerland, with over 40 attendees from 30 laboratories around the world, to agree on common solutions to the conundrum of inconsistent BMP test results. This paper presents the consensus of the intense roundtable discussions and cross-comparison of methodologies used in respective laboratories. Compulsory elements for the validation of BMP results were defined. They include the minimal number of replicates, the request to carry out blank and positive control assays, a criterion for the test duration, details on BMP calculation, and last but not least criteria for rejection of the BMP tests. Finally, recommendations on items that strongly influence the outcome of BMP tests such as inoculum characteristics, substrate preparation, test setup, and data analysis are presented to increase the probability of obtaining validated and reproducible results.info:eu-repo/semantics/publishedVersio

Structural Alterations in a Component of Cytochrome c Oxidase and Molecular Evolution of Pathogenic Neisseria in Humans

Three closely related bacterial species within the genus Neisseria are of importance to human disease and health. Neisseria meningitidis is a major cause of meningitis, while Neisseria gonorrhoeae is the agent of the sexually transmitted disease gonorrhea and Neisseria lactamica is a common, harmless commensal of children. Comparative genomics have yet to yield clear insights into which factors dictate the unique host-parasite relationships exhibited by each since, as a group, they display remarkable conservation at the levels of nucleotide sequence, gene content and synteny. Here, we discovered two rare alterations in the gene encoding the CcoP protein component of cytochrome cbb3 oxidase that are phylogenetically informative. One is a single nucleotide polymorphism resulting in CcoP truncation that acts as a molecular signature for the species N. meningitidis. We go on to show that the ancestral ccoP gene arose by a unique gene duplication and fusion event and is specifically and completely distributed within species of the genus Neisseria. Surprisingly, we found that strains engineered to express either of the two CcoP forms conditionally differed in their capacity to support nitrite-dependent, microaerobic growth mediated by NirK, a nitrite reductase. Thus, we propose that changes in CcoP domain architecture and ensuing alterations in function are key traits in successive, adaptive radiations within these metapopulations. These findings provide a dramatic example of how rare changes in core metabolic proteins can be connected to significant macroevolutionary shifts. They also show how evolutionary change at the molecular level can be linked to metabolic innovation and its reversal as well as demonstrating how genotype can be used to infer alterations of the fitness landscape within a single host

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice.

Pathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems