Comparison between Different Methods for Biomechanical Assessment of Ex Vivo Fracture Callus Stiffness in Small Animal Bone Healing Studies

For ex vivo measurements of fracture callus stiffness in small animals, different test methods, such as torsion or bending tests, are established. Each method provides advantages and disadvantages, and it is still debated which of those is most sensitive to experimental conditions (i.e. specimen alignment, directional dependency, asymmetric behavior). The aim of this study was to experimentally compare six different testing methods regarding their robustness against experimental errors. Therefore, standardized specimens were created by selective laser sintering (SLS), mimicking size, directional behavior, and embedding variations of respective rat long bone specimens. For the latter, five different geometries were created which show shifted or tilted specimen alignments. The mechanical tests included three-point bending, four-point bending, cantilever bending, axial compression, constrained torsion, and unconstrained torsion. All three different bending tests showed the same principal behavior. They were highly dependent on the rotational direction of the maximum fracture callus expansion relative to the loading direction (creating experimental errors of more than 60%), however small angular deviations (<15°) were negligible. Differences in the experimental results between the bending tests originate in their respective location of maximal bending moment induction. Compared to four-point bending, three-point bending is easier to apply on small rat and mouse bones under realistic testing conditions and yields robust measurements, provided low variation of the callus shape among the tested specimens. Axial compressive testing was highly sensitive to embedding variations, and therefore cannot be recommended. Although it is experimentally difficult to realize, unconstrained torsion testing was found to be the most robust method, since it was independent of both rotational alignment and embedding uncertainties. Constrained torsional testing showed small errors (up to 16.8%, compared to corresponding alignment under unconstrained torsion) due to a parallel offset between the specimens’ axis of gravity and the torsional axis of rotation.     

The heterotrimeric G protein G(i3) regulates hepatic autophagy downstream of the insulin receptor

Compelling evidence suggests that the heterotrimeric G protein G(i3) specifically transmits the antiautophagic effects of insulin and amino acids in the liver. This points to a previously unrecognized cross talk between the insulin receptor tyrosine kinase and G(i3). Interestingly, G(i3) is localized not only to plasma membranes but also to membranes of the autophagosomal compartment. Furthermore, as part of insulin's or phenylalanine's actions to inhibit autophagy, G(i3) is redistributed away from autophagosomes. Therefore, endomembrane-associated rather than plasma membrane-localized G(i3) may serve as the target of insulin's endocrine and metabolic actions. We therefore propose that the function and regulation of organelle-associated heterotrimeric G proteins may be different from their roles at the plasma membrane where they act as signal transducers of seven-transmembrane receptors. Here, we discuss recent findings and propose a function for G(i3) in mTOR-dependent signaling pathways. We hypothesize that G(i) family members may have tissue-specific roles in the regulation of autophagy under different physiological and pathological conditions.     

Succulent plants on arid inselbergs

Succulent plants in arid areas are believed to be often associated with high altitudes, except where they form the dominant, zonal vegetation such as in the Succulent Karoo Biome of southern Africa. To test this hypothesis the contribution of perennial succulents to inselberg floras was investigated on twenty-two isolated mountains (inselbergs) at four study sites (Etendeka–Barab: 19°34′ S/13°42′ E; Spitzkoppe: 21°49′ S/15°09′ E; Namibrand: 25°17′ S/15°47′ E; and Sperrgebiet: 27°43′ S/16°04′ E) along a nearly 1000 km bioclimatic gradient in the Namib Desert and adjacent areas, from the Succulent Karoo in the south to the northern Namib, and along altitude gradients.The contribution of succulents to the flora clearly increased with altitude at the two central study sites, thus showing a hump-shaped trend along the bioclimatic gradient. No such correlation was found at the northern and southern end of the bioclimatic gradient. The surrounding (rocky) lowlands likely resulted in a more even distribution of succulents throughout the landscape in the north, while the position in the heartland of the succulent flora in southern Africa at the southern-most site, levelled out potential patterns related to altitude. Nevertheless, even here, a fair number of succulents restricted to mountain habitats occur. This is the case in all other study sites, where succulents restricted to mountain habitats are always present.Some perennial succulents, such as Aridaria noctiflora, Euphorbia gummifera and Tetragonia reduplicata indicate an upward movement with increasing distance from the Succulent Karoo, by growing on plains and lowlands in the Succulent Karoo, but only on inselbergs at study sites north of the Succulent Karoo.Many of the mountain specialist succulents are sought after by plant collectors, such as species of Conophytum, Crassula and Lithops. Protecting and monitoring vulnerable populations as well as growing these in horticulture may help to alleviate the pressure on some of these rare species in nature. Preserving genetic material of isolated populations from mountain tops ex situ is another important conservation measure, and particularly important in the view of anticipated climatic change.     

Discovery of A-type procyanidin dimers in yellow raspberries by untargeted metabolomics and correlation based data analysis

Introduction

Raspberries are becoming increasingly popular due to their reported health beneficial properties. Despite the presence of only trace amounts of anthocyanins, yellow varieties seems to show similar or better effects in comparison to conventional raspberries.

Objectives

The aim of this work is to characterize the metabolic differences between red and yellow berries, focussing on the compounds showing a higher concentration in yellow varieties.

Methods

The metabolomic profile of 13 red and 12 yellow raspberries (of different varieties, locations and collection dates) was determined by UPLC–TOF-MS. A novel approach based on Pearson correlation on the extracted ion chromatograms was implemented to extract the pseudospectra of the most relevant biomarkers from high energy LC–MS runs. The raw data will be made publicly available on MetaboLights (MTBLS333).

Results

Among the metabolites showing higher concentration in yellow raspberries it was possible to identify a series of compounds showing a pseudospectrum similar to that of A-type procyanidin polymers. The annotation of this group of compounds was confirmed by specific MS/MS experiments and performing standard injections.

Conclusions

In berries lacking anthocyanins the polyphenol metabolism might be shifted to the formation of a novel class of A-type procyanidin polymers.

     

Spatial scales of bacterial diversity in cold-water coral reef ecosystems

Background

Cold-water coral reef ecosystems are recognized as biodiversity hotspots in the deep sea, but insights into their associated bacterial communities are still limited. Deciphering principle patterns of bacterial community variation over multiple spatial scales may however prove critical for a better understanding of factors contributing to cold-water coral reef stability and functioning.

Methodology/Principal Findings

Bacterial community structure, as determined by Automated Ribosomal Intergenic Spacer Analysis (ARISA), was investigated with respect to (i) microbial habitat type and (ii) coral species and color, as well as the three spatial components (iii) geomorphologic reef zoning, (iv) reef boundary, and (v) reef location. Communities revealed fundamental differences between coral-generated (branch surface, mucus) and ambient microbial habitats (seawater, sediments). This habitat specificity appeared pivotal for determining bacterial community shifts over all other study levels investigated. Coral-derived surfaces showed species-specific patterns, differing significantly between Lophelia pertusa and Madrepora oculata, but not between L. pertusa color types. Within the reef center, no community distinction corresponded to geomorphologic reef zoning for both coral-generated and ambient microbial habitats. Beyond the reef center, however, bacterial communities varied considerably from local to regional scales, with marked shifts toward the reef periphery as well as between different in- and offshore reef sites, suggesting significant biogeographic imprinting but weak microbe-host specificity.

Conclusions/Significance

This study presents the first multi-scale survey of bacterial diversity in cold-water coral reefs, spanning a total of five observational levels including three spatial scales. It demonstrates that bacterial communities in cold-water coral reefs are structured by multiple factors acting at different spatial scales, which has fundamental implications for the monitoring of microbial diversity and function in those ecosystems.     

Assessing sub-seafloor microbial activity by combined stable isotope probing with deuterated water and 13C-bicarbonate

Sub‐seafloor sediments are populated by large numbers of microbial cells but not much is known about their metabolic activities, growth rates and carbon assimilation pathways. Here we introduce a new method enabling the sensitive detection of microbial lipid production and the distinction of auto‐ and heterotrophic carbon assimilation. Application of this approach to anoxic sediments from a Swedish fjord allowed to compare the activity of different functional groups, the growth and turnover times of the bacterial and archaeal communities. The assay involves dual stable isotope probing (SIP) with deuterated water (D₂O) and ¹³C_DIC (d issolved i norganic c arbon). Culture experiments confirmed that the D content in newly synthesized lipids is in equilibrium with the D content in labelled water, independent of whether the culture grew hetero‐ or autotrophically. The ratio of ¹³C_DIC to D₂O incorporation enables distinction between these two carbon pathways in studies of microbial cultures and in environmental communities. Furthermore, D₂O‐SIP is sufficiently sensitive to detect the formation of few hundred cells per day in a gram of sediment. In anoxic sediments from a Swedish fjord, we found that > 99% of newly formed lipids were attributed to predominantly heterotrophic bacteria. The production rate of bacterial lipids was highest in the top 5 cm and decreased 60‐fold below this depth while the production rate of archaeal lipids was rather low throughout the top meter of seabed. The contrasting patterns in the rates of archaeal and bacterial lipid formation indicate that the factors controlling the presence of these two lipid groups must differ fundamentally.     

Beta-arrestin binding to CC chemokine receptor 5 requires multiple C-terminal receptor phosphorylation sites and involves a conserved Asp-Arg-Tyr sequence motif

Agonist binding to the CC chemokine receptor 5 (CCR5) induces the phosphorylation of four distinct serine residues that are located in the CCR5 C terminus. We established a series of clonal RBL-2H3 cell lines expressing CCR5 with alanine mutations of Ser(336), Ser(337), Ser(342), and Ser(349) in various combinations and explored the significance of phosphorylation sites for the ability of the receptor to interact with beta-arrestins and to undergo desensitization and internalization upon ligand binding. Receptor mutants that lack any two phosphorylation sites retained their ability to recruit endogenous beta-arrestins to the cell membrane and were normally sequestered, whereas alanine mutation of any three C-terminal serine residues abolished both beta-arrestin binding and rapid agonist-induced internalization. In contrast, RANTES (regulated on activation normal T cell expressed and secreted) stimulation of a S336A/S349A mutant triggered a sustained calcium response and enhanced granular enzyme release. This mutational analysis implies that CCR5 internalization largely depends on a beta-arrestin-mediated mechanism that requires the presence of any two phosphorylation sites, whereas receptor desensitization is independently regulated by the phosphorylation of distinct serine residues. Surface plasmon resonance analysis further demonstrated that purified beta-arrestin 1 binds to phosphorylated and nonphosphorylated C-tail peptides with similar affinities, suggesting that beta-arrestins use additional receptor sites to discriminate between nonactivated and activated receptors. Surface plasmon resonance analysis revealed beta-arrestin 1 binding to the second intracellular loop of CCR5, which required an intact Asp-Arg-Tyr triplet. These results suggest that a conserved sequence motif within the second intracellular loop of CCR5 that is known to be involved in G protein activation plays a significant role in beta-arrestin binding to CCR5.     

PRMT1 promotes the tumor suppressor function of p14ARF and is indicative for pancreatic cancer prognosis

The p14^ARF protein is a well‐known regulator of p53‐dependent and p53‐independent tumor‐suppressive activities. In unstressed cells, p14^ARF is predominantly sequestered in the nucleoli, bound to its nucleolar interaction partner NPM. Upon genotoxic stress, p14^ARF undergoes an immediate redistribution to the nucleo‐ and cytoplasm, where it promotes activation of cell cycle arrest and apoptosis. Here, we identify p14^ARF as a novel interaction partner and substrate of PRMT1 (protein arginine methyltransferase 1). PRMT1 methylates several arginine residues in the C‐terminal nuclear/nucleolar localization sequence (NLS/NoLS) of p14^ARF. In the absence of cellular stress, these arginines are crucial for nucleolar localization of p14^ARF. Genotoxic stress causes augmented interaction between PRMT1 and p14^ARF, accompanied by arginine methylation of p14^ARF. PRMT1‐dependent NLS/NoLS methylation promotes the release of p14^ARF from NPM and nucleolar sequestration, subsequently leading to p53‐independent apoptosis. This PRMT1‐p14^ARF cooperation is cancer‐relevant and indicative for PDAC (pancreatic ductal adenocarcinoma) prognosis and chemotherapy response of pancreatic tumor cells. Our data reveal that PRMT1‐mediated arginine methylation is an important trigger for p14^ARF’s stress‐induced tumor‐suppressive function.