Thrombin Generation in Zebrafish Blood

To better understand hypercoagulability as an underlying cause for thrombosis, the leading cause of death in the Western world, new assays to study ex vivo coagulation are essential. The zebrafish is generally accepted as a good model for human hemostasis and thrombosis, as the hemostatic system proved to be similar to that in man. Their small size however, has been a hurdle for more widespread use in hemostasis related research. In this study we developed a method that enables the measurement of thrombin generation in a single drop of non-anticoagulated zebrafish blood. Pre-treatment of the fish with inhibitors of FXa and thrombin, resulted in a dose dependent diminishing of thrombin generation, demonstrating the validity of the assay. In order to establish the relationship between whole blood thrombin generation and fibrin formation, we visualized the resulting fibrin network by scanning electron microscopy. Taken together, in this study we developed a fast and reliable method to measure thrombin generation in whole blood collected from a single zebrafish. Given the similarities between coagulation pathways of zebrafish and mammals, zebrafish may be an ideal animal model to determine the effect of novel therapeutics on thrombin generation. Additionally, because of the ease with which gene functions can be silenced, zebrafish may serve as a model organism for mechanistical research in thrombosis and hemostasis.     

Cryptic species and colonization processes in Ophryotrocha (Annelida,Dorvilleidae) inhabiting vertebrate remains in the shallow‐water Mediterranean

Over the past several years, there has been growing interest in how bones of decaying mammals are colonized in the marine seabed. One of the most common opportunistic taxa occurring worldwide on bones is dorvilleid polychaetes of the genus Ophryotrocha. In a recent study in the Mediterranean, Ophryotrocha puerilis and Ophryotrocha alborana were two of the most abundant species occurring in experimentally deployed bones. These species have direct development and this makes them a suitable model to study the mechanisms and processes allowing organisms lacking a dispersive larval phase to colonize new substrates. Here, we address the colonization processes at the molecular level for populations of O. puerilis and O. alborana on experimentally deployed mammal bones in the shallow‐water Mediterranean collected over a year at 3‐month intervals. High genetic distances between some of the O. puerilis organisms collected indicated the occurrence of at least two cryptic sibling species (O. puerilis ‘Shallow’ and O. puerilis ‘Deep’) apart from O. puerilis sensu stricto. This was corroborated with phylogenetic analyses using an alignment of three concatenated genes (COI, 16S, H3) and with species delimitation analyses using COI. The haplotype network inferred from COI sequences for O. puerilis ‘Shallow’ showed a few common haplotypes shared between the two trimesters analysed and several other less represented haplotypes only present in one trimester. Thus, colonization of these experimental bones may have been achieved by a few organisms that arrived to the bones and were able to reseed, and by several individuals arriving to the experimental bones and not persisting across time. In contrast, the haplotype network for O. alborana revealed that none of the haplotypes present in three different trimesters were shared, suggesting that the populations arriving at the bones during each trimester were totally replaced by new individuals during the subsequent trimesters. Our study suggests that different species of shallow‐water Ophryotrocha occurring in the Mediterranean may have different patterns of substrate colonization despite sharing similar life histories.     

The Role of Electrostatic Interactions in Binding of Histone H3K4me2/3 to the Sgf29 Tandem Tudor Domain

Several reader domain proteins that specifically recognize methyllysine-containing histones contain the negatively-charged aspartate or glutamate residues as part of the aromatic cage. Herein, we report thermodynamic analyses for the recognition of histone H3K4me3 and H3K4me2 by the tandem tudor domain of Sgf29 and its recognition site variants. Small uncharged and large aromatic substitutions on the Asp266 site resulted in a significant decrease in binding affinities for both H3K4me3 and H3K4me2, demonstrating the role of the negative charge of Asp266 in the readout process by Sgf29. This study emphasizes the essential contribution of electrostatic interactions to the overall binding affinity, and reveals that the underlying mechanisms for the recognition of Kme2/3 depend on the composition and arrangement of the aromatic cage.     

Cardiolipin synthases of Escherichia coli have phospholipid class specific phospholipase D activity dependent on endogenous and foreign phospholipids

E. coli has three Cls-isoenzymes for cardiolipin (CL) synthesis but the differences between these three enzymes remain unresolved. All three Cls enzymes contain the phospholipase D (PLD) characteristic HKD motive and synthesize CL using PLD activity. Here, using LC-MS we show the effect of overexpressing or deletion of the three individual Cls enzymes on the lipidome, which included changes in lipid class distribution and CL species profiles. We demonstrate, for the first time, that overexpression of only ClsB resulted in the appreciable synthesis of a variety of phosphatidylalcohols, thereby establishing a ‘classic’ PLD activity for this enzyme: phospholipid headgroup exchange. Endogenous E. coli lipids and primary alcohols were substrates for this trans-phosphatidylation reaction. Furthermore, we show that endogenous levels of ClsA mediated a similar trans-phosphatidylation reaction to form phosphatidylalcohols, however this reaction was dependent on the presence of the foreign phospholipid class phosphatidylcholine (PC). This allows us to clarify the different specificities of the cardiolipin synthases.     

Vasoplegia after implantation of a continuous flow left ventricular assist device: incidence,outcomes and predictors

Background

Vasoplegia after routine cardiac surgery is associated with severe postoperative complications and increased mortality. It is also prevalent in patients undergoing implantation of pulsatile flow left ventricular assist devices (LVAD). However, less is known regarding vasoplegia after implantation of newer generations of continuous flow LVADs (cfLVAD). We aim to report the incidence, impact on outcome and predictors of vasoplegia in these patients.

Methods

Adult patients scheduled for primary cfLVAD implantation were enrolled into a derivation cohort (n?=?118, 2006–2013) and a temporal validation cohort (n?=?73, 2014–2016). Vasoplegia was defined taking into consideration low mean arterial pressure and/or low systemic vascular resistance, preserved cardiac index and high vasopressor support. Vasoplegia was considered after bypass and the first 48?h of ICU stay lasting at least three consecutive hours. This concept of vasoplegia was compared to older definitions reported in the literature in terms of the incidence of postoperative vasoplegia and its association with adverse outcomes. Logistic regression was used to identify independent predictors. Their ability to discriminate patients with vasoplegia was quantified by the area under the receiver operating characteristic curve (AUC).

Results

The incidence of vasoplegia was 33.1% using the unified definition of vasoplegia. Vasoplegia was associated with increased ICU length-of-stay (10.5 [6.9–20.8] vs 6.1 [4.6–10.4] p?=?0.002), increased ICU-mortality (OR 5.8, 95% CI 1.9–18.2) and one-year-mortality (OR 3.9, 95% CI 1.5–10.2), and a higher incidence of renal failure (OR 4.3, 95% CI 1.8–10.4). Multivariable analysis identified previous cardiothoracic surgery, preoperative dopamine administration, preoperative bilirubin levels and preoperative creatinine clearance as independent preoperative predictors of vasoplegia. The resultant prediction model exhibited a good discriminative ability (AUC 0.80, 95% CI 0.71–0.89, p?<? 0.01). Temporal validation resulted in an AUC of 0.74 (95% CI 0.61–0.87, p?<? 0.01).

Conclusions

In the era of the new generation of cfLVADs, vasoplegia remains a prevalent (33%) and critical condition with worse short-term outcomes and survival. We identified previous cardiothoracic surgery, preoperative treatment with dopamine, preoperative bilirubin levels and preoperative creatinine clearance as independent predictors.

     

Disentangling vegetation and climate as drivers of Australian vertebrate richness

Determining drivers of species richness is recognised as highly complex, involving many synergies and interactions. We examine the utility of newly available remote sensing representations of vegetation productivity and vegetation structure to examine drivers of species richness at continental and regional scales. We related richness estimates derived from stacked species distribution models for birds, mammals, amphibians, and reptiles to estimates of actual and potential evapotranspiration (AET and PET), forest structure, and forest productivity across Australia as a whole as well as by bioclimatic zones. We used structural equation modeling to partition correlations between climate energy and vegetation attributes and their subsequent associations with species richness. Continentally, vertebrate richness patterns were strongly related to patterns of energy availability. Richness of amphibians, mammals, and birds were positively associated with AET. However, reptile richness was most strongly associated with PET. Regionally, forest structure and productivity associations with bird, mammal, and amphibian richness were strongest. Again, reptile richness associated most strongly with PET. Our results suggest that a hierarchy of drivers of broad‐scale vertebrate richness patterns exist (reptiles excluded): 1) climate energy is most important at the continental scale; next, 2) vegetation productivity and vegetation structure are most important at the regional scale; except 3) at low extremes of climate energy when energy becomes limiting.     

Shellfish Reefs Increase Water Storage Capacity on Intertidal Flats Over Extensive Spatial Scales

Ecosystem engineering species can affect their environment at multiple spatial scales, from the local scale up to a significant distance, by indirectly affecting the surrounding habitats. Structural changes in the landscape can have important consequences for ecosystem functioning, for example, by increasing retention of limiting resources in the system. Yet, it remains poorly understood how extensive the footprint of ecosystem engineers on the landscape is. Using remote sensing techniques, we reveal that depression storage capacity on intertidal flats is greatly enhanced by engineering by shellfish resulting in intertidal pools. Many organisms use such pools to bridge low water events. This storage capacity was significantly higher both locally within the shellfish reef, but also at extensive spatial scales up to 115 m beyond the physical reef borders. Therefore, the footprint of these ecosystem engineers on the landscape was more than 5 times larger than their actual coverage; the shellfish cover approximately 2% of the total intertidal zone, whereas they influence up to approximately 11% of the area by enhancing water storage capacity. We postulate that increased residence time of water due to higher water storage capacity within engineered landscapes is an important determinant of ecosystem functioning that may extend well beyond the case of shellfish reefs provided here.