Phenols in reproductive and somatic structures of lichens: a case of optimal defence?

Optimal defence theory (ODT) attempts to explain variation in plant secondary compounds between different species, different growth conditions and different parts of individual plants. The theory is widely applied to vascular plants and more recently also to seaweeds. Surprisingly, ODT has gained little attention as potential explanation on the distribution of lichen secondary metabolites. In the present study, we analysed intrathalline variation in total phenol content and phenol spectra between reproductive and somatic structures of three foliose lichens, Xanthoria parietina , Vulpicida pinastri and Hypogymnia physodes . The results showed that the concentration of phenolic compounds is higher in sorediate than in non-sorediate lobe ends of V. pinastri and H. physodes as well as in apothecia of X. parietina compared to other parts of the thallus. These results were in accordance with ODT predicting higher allocation of phenols in structures that are most important for the fitness of an individual genet or ramet. This pattern was parallel in all species regardless whether the compounds originate from either acetate-mevalonate or shikimic acid pathways. Moreover, both sexual ( X. parietina apothecia) and asexual (soralia of V. pinastri and H. physodes ) reproductive structures were higher in phenols compared to somatic tissue.     

In vivo homopropargylglycine incorporation enables sampling,isolation and characterization of nascent proteins from Arabidopsis thaliana

Determining which proteins are actively synthesized at a given point in time and extracting a representative sample for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) of a small sample of the proteins being synthesized in Arabidopsis plants or cell cultures, facilitating their click-chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met sites throughout protein amino acid sequences and correlation with independent studies of protein labelling with ¹⁵N verified the data. We provide evidence that HPG-based BONCAT tags a better sample of nascent plant proteins than azidohomoalanine (AHA)-based BONCAT in Arabidopsis and show that the AHA induction of Met metabolism and greater inhibition of cell growth rate than HPG probably limits AHA incorporation at Met sites in Arabidopsis. We show HPG-based BONCAT provides a verifiable method for sampling, which plant proteins are being synthesized at a given time point and enriches a small portion of new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides samples were found to contain significantly fewer common post-translationally modified residues than the same proteins from whole plant extracts, providing evidence for age-related accumulation of post-translational modifications in plants.     

Faecal Metaproteomic Analysis Reveals a Personalized and Stable Functional Microbiome and Limited Effects of a Probiotic Intervention in Adults

Recent metagenomic studies have demonstrated that the overall functional potential of the intestinal microbiome is rather conserved between healthy individuals. Here we assessed the biological processes undertaken in-vivo by microbes and the host in the intestinal tract by conducting a metaproteome analysis from a total of 48 faecal samples of 16 healthy adults participating in a placebo-controlled probiotic intervention trial. Half of the subjects received placebo and the other half consumed Lactobacillus rhamnosus GG for three weeks (10¹⁰ cfu per day). Faecal samples were collected just before and at the end of the consumption phase as well as after a three-week follow-up period, and were processed for microbial composition and metaproteome analysis. A common core of shared microbial protein functions could be identified in all subjects. Furthermore, we observed marked differences in expressed proteins between subjects that resulted in the definition of a stable and personalized microbiome both at the mass-spectrometry-based proteome level and the functional level based on the KEGG pathway analysis. No significant changes in the metaproteome were attributable to the probiotic intervention. A detailed taxonomic assignment of peptides and comparison to phylogenetic microarray data made it possible to evaluate the activity of the main phyla as well as key species, including Faecalibacterium prausnitzii. Several correlations were identified between human and bacterial proteins. Proteins of the human host accounted for approximately 14% of the identified metaproteome and displayed variations both between and within individuals. The individually different human intestinal proteomes point to personalized host-microbiota interactions. Our findings indicate that analysis of the intestinal metaproteome can complement gene-based analysis and contributes to a thorough understanding of the activities of the microbiome and the relevant pathways in health and disease.     

Manually and automatically produced pellet cultures of human primary chondrocytes: A comparative analysis

Cartilage defects are often associated with restriction of the locomotor system. New methods are required to investigate cartilage tissue and for the repair of cartilage tissue. 3D cultures are promising due to better simulation of in vivo conditions. The aim of this study was to provide a model system for studying cartilage tissue. We solved this problem by automated production of pellet cultures of human primary chondrocytes in media with and without antibiotics using the Biomek® Cell Workstation and consequent automated bioscreening with a high‐throughput screening system, and compared with the regular manual processes. The Biomek® Cell Workstation allows the cultivation of different cell types (suspensions cells and adherent cells) and 3D cell cultures (pellet cultures, alginate beads and spheroid cultures). The proliferation was analyzed by DNA quantification and compared with the EZ4U proliferation assay as a new tool for pellet cultures. The toxicity was evaluated by the detection of ubiquitous adenylate kinases. The proliferation increased from day 14 until day 35 and was associated with a decrease in the cytotoxicity. The comparative analysis showed similar results for manual and automated processes. We concluded that the manual methods can be replaced by automated processes (pellet manufacturing and screening), which would allow large‐scale procedures to support studies on cartilage regeneration.     

Shotgun metagenomics of soil invertebrate communities reflects taxonomy,biomass, and reference genome properties

• Metagenomics – shotgun sequencing of all DNA fragments from a community DNA extract – is routinely used to describe the composition, structure, and function of microorganism communities. Advances in DNA sequencing and the availability of genome databases increasingly allow the use of shotgun metagenomics on eukaryotic communities. Metagenomics offers major advances in the recovery of biomass relationships in a sample, in comparison to taxonomic marker gene‐based approaches (metabarcoding). However, little is known about the factors which influence metagenomics data from eukaryotic communities, such as differences among organism groups, the properties of reference genomes, and genome assemblies.
• We evaluated how shotgun metagenomics records composition and biomass in artificial soil invertebrate communities at different sequencing efforts. We generated mock communities of controlled biomass ratios from 28 species from all major soil mesofauna groups: mites, springtails, nematodes, tardigrades, and potworms. We shotgun sequenced these communities and taxonomically assigned them with a database of over 270 soil invertebrate genomes.
• We recovered over 95% of the species, and observed relatively high false‐positive detection rates. We found strong differences in reads assigned to different taxa, with some groups (e.g., springtails) consistently attracting more hits than others (e.g., enchytraeids). Original biomass could be predicted from read counts after considering these taxon‐specific differences. Species with larger genomes, and with more complete assemblies, consistently attracted more reads than species with smaller genomes. The GC content of the genome assemblies had no effect on the biomass–read relationships. Results were similar among different sequencing efforts.
• The results show considerable differences in taxon recovery and taxon specificity of biomass recovery from metagenomic sequence data. The properties of reference genomes and genome assemblies also influence biomass recovery, and they should be considered in metagenomic studies of eukaryotes. We show that low‐ and high‐sequencing efforts yield similar results, suggesting high cost‐efficiency of metagenomics for eukaryotic communities. We provide a brief roadmap for investigating factors which influence metagenomics‐based eukaryotic community reconstructions. Understanding these factors is timely as accessibility of DNA sequencing and momentum for reference genomes projects show a future where the taxonomic assignment of DNA from any community sample becomes a reality.

     

Bone morphogenetic proteins 4 and 2/7 induce osteogenic differentiation of mouse skin derived fibroblast and dermal papilla cells

Heterotopic ossification is a pathological condition in which bone forms outside the skeletal system. It can also occur in skin, which is the case in some genetic disorders. In addition to precursor cells and the appropriate tissue environment, heterotopic ossification requires inductive signals such as bone morphogenetic proteins (BMP). BMPs are growth and differentiation factors that have the ability to induce cartilage and bone formation in ectopic sites. The objective of this study is to explore the effect of the BMP-4 homodimer and BMP-2/7 heterodimer on the osteogenic differentiation of primary mouse skin fibroblasts and hair follicle dermal papilla (DP) cells. Osteogenic differentiation was induced by osteogenic induction medium (OS) containing 10 nM dexamethasone. The effect of BMP-4 and BMP-2/7 was studied using alkaline phosphatase (ALP) and calcium assays after 1.5, 3 and 5 weeks of differentiation. Fibroblasts and DP cells were able to differentiate into osteoblast-like matrix mineralizing cells. The first visible sign of differentiation was the change of morphology from rounded to more spindle-shaped cells. BMP-4 and BMP-2/7 exposure elevated ALP activity and calcium production significantly more than OS alone. The osteogenic response to BMP-4 and BMP-2/7 was similar in fibroblasts, whereas, in DP cells, BMP-2/7 was more potent than BMP-4. OS alone could not induce osteogenic differentiation in DP cells. Clear and consistent results show that dermal fibroblasts and stem cells from the dermal papilla were capable of osteogenic differentiation. The BMP-2/7 heterodimer was significantly more effective on hair follicular dermal stem cell differentiation.