Multiple Labeling in Electron Microscopy: Its Application in Cardiovascular Research

The heart is a muscular pump kept together by a network of extracellular matrix components. An increase in collagens, as in chronic congestive heart failure (CHF), is thought to have a negative effect on cardiac compliance and, thus, on the clinical condition. Conventional electron microscopy allows for the study of cellular and extracellular components and scanning electron microscopy (SEM) can put these structures in three-dimensional perspective. However, in order to study extracellular matrix components in relation to cells, immunoelectron microscopy is superior. We have used this technique in our studies on heart failure. Heart specimens were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in sodium cacodylate buffer, dehydrated by the method of progressive lowering of temperature and embedded in LR Gold plastic. Immunolabeling could be achieved with different sized gold-conjugated secondary antibodies or protein-A gold conjugates. Depending on the objective, ultra small gold (USG) conjugates or a regular probe size can be used. Labeling efficiency could be increased by bridging antibodies. The double and triple staining procedures were based on single staining methods using one-and two-face labeling. The choice of antibodies and gold conjugates depended on the objectives. Immunoelectron microscopy, using multiple labeling, allowed a detailed study of the organization of the extracellular matrix and its relationship with cardiac myocytes. This may prove to be a useful tool for the study of chronic heart failure

Diversity and activity of marine bacterioplankton during a diatom bloom in the North Sea assessed by total RNA and pyrotag sequencing

A recent investigation of bacterioplankton communities in the German Bight towards the end of a diatomdominated spring phytoplankton bloom revealed pronounced successions of distinct bacterial clades. A combination of metagenomics and metaproteomics indicated that these clades had distinct substrate spectra and consumed different algal substrates. In this study we re-analyzed samples from the initial study by total community RNA (metatranscriptomics) and 16S rRNA gene amplicon sequencing. This complementary approach provided new insights into the community composition and expressed genes as well as the assessment of metabolic activity levels of distinct clades. Flavobacteria (genera Ulvibacter, Formosa, and Polaribacter), Alphaproteobacteria (SARI 1 clade and Rhodobacteraceae) and Gammaproteobacteria (genus Reinekea and SAR92 dade) were the most abundant taxa. Mapping of the metatranscriptome data on assembled and taxonomically classified metagenome data of the same samples substantiated that Formosa and Polaribacter acted as major algal polymer degraders, whereas Rhodobacteraceae and Reinekea spp. exhibited less specialized substrate spectra. In addition, we found that members of the Rhodobacteraceae and SAR92 clade showed high metabolic activity levels, which suggests that these clades played a more important role during the bloom event as indicated by their in situ abundances. (c) 2014 The Authors. Published by Elsevier B.V

Niche differentiation among annually recurrent coastal marine group II Euryarchaeota

Since the discovery of archaeoplankton in 1992, the euryarchaeotal Marine Group II (MGII) remains uncultured and less understood than other planktonic archaea. We characterized the seasonal dynamics of MGII populations in the southern North Sea on a genomic and microscopic level over the course of four years. We recovered 34 metagenome-assembled genomes (MAGs) of MGIIa and MGIIb that corroborated proteorhodopsin-based photoheterotrophic lifestyles. However, MGIIa and MGIIb MAG genome sizes differed considerably (~1.9 vs. ~1.4 Mbp), as did their transporter, peptidase, flagella and sulfate assimilation gene repertoires. MGIIb populations were characteristic of winter samples, whereas MGIIa accounted for up to 23% of the community at the beginning of summer. Both clades consisted of annually recurring, sequence-discrete populations with low intra-population sequence diversity. Oligotyping of filtered cell-size fractions and microscopy consistently suggested that MGII cells were predominantly free-living. Cells were coccoid and ~0.7 µm in diameter, likely resulting in grazing avoidance. Based on multiple lines of evidence, we propose distinct niche adaptations of MGIIa and MGIIb Euryarchaeota populations that are characteristic of summer and winter conditions in the coastal North Sea

Functional characterization of polysaccharide utilization loci in the marine Bacteroidetes 'Gramella forsetii' KT0803

Members of the phylum Bacteroidetes are abundant in many marine ecosystems and are known to have a pivotal role in the mineralization of complex organic substrates such as polysaccharides and proteins. We studied the decomposition of the algal glycans laminarin and alginate by 'Gramella forsetii' KT0803, a bacteroidetal isolate from North Sea surface waters. A combined application of isotope labeling, subcellular protein fractionation and quantitative proteomics revealed two large polysaccharide utilization loci (PULs) that were specifically induced, one by alginate and the other by laminarin. These regulons comprised genes of surface-exposed proteins such as oligomer transporters, substrate-binding proteins, carbohydrate-active enzymes and hypothetical proteins. Besides, several glycan-specific TonB-dependent receptors and SusD-like substrate-binding proteins were expressed also in the absence of polysaccharide substrates, suggesting an anticipatory sensing function. Genes for the utilization of the beta-1,3-glucan laminarin were found to be co-regulated with genes for glucose and alpha-1,4-glucan utilization, which was not the case for the non-glucan alginate. Strong syntenies of the PULs of 'G. forsetii' with similar loci in other Bacteroidetes indicate that the specific response mechanisms of 'G. forsetii' to changes in polysaccharide availability likely apply to other Bacteroidetes. Our results can thus contribute to an improved understanding of the ecological niches of marine Bacteroidetes and their roles in the polysaccharide decomposition part of carbon cycling in marine ecosystems

Analysis and comparison of very large metagenomes with fast clustering and functional annotation

<p>Abstract</p> <p>Background</p> <p>The remarkable advance of metagenomics presents significant new challenges in data analysis. Metagenomic datasets (metagenomes) are large collections of sequencing reads from anonymous species within particular environments. Computational analyses for very large metagenomes are extremely time-consuming, and there are often many novel sequences in these metagenomes that are not fully utilized. The number of available metagenomes is rapidly increasing, so fast and efficient metagenome comparison methods are in great demand.</p> <p>Results</p> <p>The new metagenomic data analysis method Rapid Analysis of Multiple Metagenomes with a Clustering and Annotation Pipeline (<b>RAMMCAP</b>) was developed using an ultra-fast sequence clustering algorithm, fast protein family annotation tools, and a novel statistical metagenome comparison method that employs a unique graphic interface. RAMMCAP processes extremely large datasets with only moderate computational effort. It identifies raw read clusters and protein clusters that may include novel gene families, and compares metagenomes using clusters or functional annotations calculated by RAMMCAP. In this study, RAMMCAP was applied to the two largest available metagenomic collections, the "Global Ocean Sampling" and the "Metagenomic Profiling of Nine Biomes".</p> <p>Conclusion</p> <p>RAMMCAP is a very fast method that can cluster and annotate one million metagenomic reads in only hundreds of CPU hours. It is available from <url>http://tools.camera.calit2.net/camera/rammcap/</url>.</p

Glycoside hydrolase from the GH76 family indicates that marine Salegentibacter sp. Hel_I_6 consumes alpha-mannan from fungi

Microbial glycan degradation is essential to global carbon cycling. The marine bacterium Salegentibacter sp. Hel_I_6 (Bacteroidota) isolated from seawater off Helgoland island (North Sea) contains an α-mannan inducible gene cluster with a GH76 family endo-α-1,6-mannanase (ShGH76). This cluster is related to genetic loci employed by human gut bacteria to digest fungal α-mannan. Metagenomes from the Hel_I_6 isolation site revealed increasing GH76 gene frequencies in free-living bacteria during microalgae blooms, suggesting degradation of α-1,6-mannans from fungi. Recombinant ShGH76 protein activity assays with yeast α-mannan and synthetic oligomannans showed endo-α-1,6-mannanase activity. Resolved structures of apo-ShGH76 (2.0 Å) and of mutants co-crystalized with fungal mannan-mimicking α-1,6-mannotetrose (1.90 Å) and α-1,6-mannotriose (1.47 Å) retained the canonical (α/α)6 fold, despite low identities with sequences of known GH76 structures (GH76s from gut bacteria: <27%). The apo-form active site differed from those known from gut bacteria, and co-crystallizations revealed a kinked oligomannan conformation. Co-crystallizations also revealed precise molecular-scale interactions of ShGH76 with fungal mannan-mimicking oligomannans, indicating adaptation to this particular type of substrate. Our data hence suggest presence of yet unknown fungal α-1,6-mannans in marine ecosystems, in particular during microalgal blooms

Melatonin Alters Age-Related Changes in Transcription Factors and Kinase Activation

Male mice were fed 40 ppm melatonin for 2 months prior to sacrifice at age 26 months, and compared with both 26 and 4 month-old untreated controls. The nuclear translocation of NF-κB increased with age in both brain and spleen and this was reversed by melatonin only in brain. Another transcription factor, AP-1 was increased with age in the spleen and not in brain and this could be blocked by melatonin treatment. The fraction of the active relative to the inactive form of several enabling kinases was compared. The proportion of activated ERK was elevated with age in brain and spleen but this change was unresponsive to melatonin. A similar age-related increase in glial fibrillary acidic protein (GFAP) was also refractory to melatonin treatment. The cerebral melatonin M1 receptor decreased with age in brain but increased in spleen. The potentially beneficial nature of melatonin for the preservation of brain function with aging was suggested by the finding that an age-related decline in cortical synaptophysin levels was prevented by dietary melatonin

A quantitative account of genomic island acquisitions in prokaryotes

<p>Abstract</p> <p>Background</p> <p>Microbial genomes do not merely evolve through the slow accumulation of mutations, but also, and often more dramatically, by taking up new DNA in a process called horizontal gene transfer. These innovation leaps in the acquisition of new traits can take place via the introgression of single genes, but also through the acquisition of large gene clusters, which are termed Genomic Islands. Since only a small proportion of all the DNA diversity has been sequenced, it can be hard to find the appropriate donors for acquired genes via sequence alignments from databases. In contrast, relative oligonucleotide frequencies represent a remarkably stable genomic signature in prokaryotes, which facilitates compositional comparisons as an alignment-free alternative for phylogenetic relatedness.</p> <p>In this project, we test whether Genomic Islands identified in individual bacterial genomes have a similar genomic signature, in terms of relative dinucleotide frequencies, and can therefore be expected to originate from a common donor species.</p> <p>Results</p> <p>When multiple Genomic Islands are present within a single genome, we find that up to 28% of these are compositionally very similar to each other, indicative of frequent recurring acquisitions from the same donor to the same acceptor.</p> <p>Conclusions</p> <p>This represents the first quantitative assessment of common directional transfer events in prokaryotic evolutionary history. We suggest that many of the resident Genomic Islands per prokaryotic genome originated from the same source, which may have implications with respect to their regulatory interactions, and for the elucidation of the common origins of these acquired gene clusters.</p

Haemoglobin causes neuronal damage in vivo which is preventable by haptoglobin

After subarachnoid haemorrhage, prolonged exposure to toxic extracellular haemoglobin occurs in the brain. Here, we investigate the role of haemoglobin neurotoxicity in vivo and its prevention. In humans after subarachnoid haemorrhage, haemoglobin in cerebrospinal fluid was associated with neurofilament light chain, a marker of neuronal damage. Most haemoglobin was not complexed with haptoglobin, an endogenous haemoglobin scavenger present at very low concentration in the brain. Exogenously added haptoglobin bound most uncomplexed haemoglobin, in the first 2 weeks after human subarachnoid haemorrhage, indicating a wide therapeutic window. In mice, the behavioural, vascular, cellular and molecular changes seen after human subarachnoid haemorrhage were recapitulated by modelling a single aspect of subarachnoid haemorrhage: prolonged intrathecal exposure to haemoglobin. Haemoglobin-induced behavioural deficits and astrocytic, microglial and synaptic changes were attenuated by haptoglobin. Haptoglobin treatment did not attenuate large-vessel vasospasm, yet improved clinical outcome by restricting diffusion of haemoglobin into the parenchyma and reducing small-vessel vasospasm. In summary, haemoglobin toxicity is of clinical importance and preventable by haptoglobin, independent of large-vessel vasospasm