Discrepant Fibrinolytic Response in Plasma and Whole Blood during Experimental Endotoxemia in Healthy Volunteers

Background

Sepsis induces early activation of coagulation and fibrinolysis followed by late fibrinolytic shutdown and progressive endothelial damage. The aim of the present study was to investigate and compare the functional hemostatic response in whole blood and plasma during experimental human endotoxemia by the platelet function analyzer, Multiplate and by standard and modified thrombelastography (TEG).

Methods

Prospective physiologic study of nine healthy male volunteers undergoing endotoxemia by means of a 4-hour infusion of E. coli lipopolysaccharide (LPS, 0.5 ng/kg/hour), with blood sampled at baseline and at 4 h and 6 h. Physiological and standard biochemical data and coagulation tests, TEG (whole blood: TEG, heparinase-TEG, Functional Fibrinogen; plasma: TEG±tissue-type plasminogen activator (tPA)) and Multiplate (TRAPtest, ADPtest, ASPItest, COLtest) were recorded. Mixed models with Tukey post hoc tests and correlations were applied.

Results

Endotoxemia induced acute SIRS with increased HR, temperature, WBC, CRP and procalcitonin and decreased blood pressure. It also induced a hemostatic response with platelet consumption and reduced APTT while INR increased (all p<0.05). Platelet aggregation decreased (all tests, p<0.05), whereas TEG whole blood clot firmness increased (G, p = 0.05). Furthermore, during endotoxemia (4 h), whole blood fibrinolysis increased (clot lysis time (CLT), p<0.001) and Functional Fibrinogen clot strength decreased (p = 0.049). After endotoxemia (6 h), whole blood fibrinolysis was reduced (CLT, p<0.05). In contrast to findings in whole blood, the plasma fibrin clot became progressively more resistant towards tPA-induced fibrinolysis at both 4 h and 6 h (p<0.001).

Conclusions

Endotoxemia induced a hemostatic response with reduced primary but enhanced secondary hemostasis, enhanced early fibrinolysis and fibrinogen consumption followed by downregulation of fibrinolysis, with a discrepant fibrinolytic response in plasma and whole blood. The finding that blood cells are critically involved in the vasculo-fibrinolytic response to acute inflammation is important given that disturbances in the vascular system contribute significantly to morbidity and mortality in critically ill patients.     

Massive Sorghum Collection Genotyped with SSR Markers to Enhance Use of Global Genetic Resources

Large ex situ collections require approaches for sampling manageable amounts of germplasm for in-depth characterization and use. We present here a large diversity survey in sorghum with 3367 accessions and 41 reference nuclear SSR markers. Of 19 alleles on average per locus, the largest numbers of alleles were concentrated in central and eastern Africa. Cultivated sorghum appeared structured according to geographic regions and race within region. A total of 13 groups of variable size were distinguished. The peripheral groups in western Africa, southern Africa and eastern Asia were the most homogeneous and clearly differentiated. Except for Kafir, there was little correspondence between races and marker-based groups. Bicolor, Caudatum, Durra and Guinea types were each dispersed in three groups or more. Races should therefore better be referred to as morphotypes. Wild and weedy accessions were very diverse and scattered among cultivated samples, reinforcing the idea that large gene-flow exists between the different compartments. Our study provides an entry to global sorghum germplasm collections. Our reference marker kit can serve to aggregate additional studies and enhance international collaboration. We propose a core reference set in order to facilitate integrated phenotyping experiments towards refined functional understanding of sorghum diversity.     

The Advocacy for Pedestrian Safety Study: Cluster Randomised Trial Evaluating a Political Advocacy Approach to Reduce Pedestrian Injuries in Deprived Communities

Objective

To determine whether advocacy targeted at local politicians leads to action to reduce the risk of pedestrian injury in deprived areas.

Design

Cluster randomised controlled trial.

Setting

239 electoral wards in 57 local authorities in England and Wales.

Participants

617 elected local politicians.

Interventions

Intervention group politicians were provided with tailored information packs, including maps of casualty sites, numbers injured and a synopsis of effective interventions.

Main outcome measures

25–30 months post intervention, primary outcomes included: electoral ward level: percentage of road traffic calmed; proportion with new interventions; school level: percentage with 20 mph zones, Safe Routes to School, pedestrian training or road safety education; politician level: percentage lobbying for safety measures. Secondary outcomes included politicians’ interest and involvement in injury prevention, and facilitators and barriers to implementation.

Results

Primary outcomes did not significantly differ: % difference in traffic calming (0.07, 95%CI: −0.07 to 0.20); proportion of schools with 20 mph zones (RR 1.47, 95%CI: 0.93 to 2.32), Safe Routes to School (RR 1.34, 95%CI: 0.83 to 2.17), pedestrian training (RR 1.23, 95%CI: 0.95 to 1.61) or other safety education (RR 1.16, 95%CI: 0.97 to 1.39). Intervention group politicians reported greater interest in child injury prevention (RR 1.09, 95%CI 1.03 to 1.16), belief in potential to help prevent injuries (RR 1.36, 95%CI 1.16 to 1.61), particularly pedestrian safety (RR 1.55, 95%CI 1.19 to 2.03). 63% of intervention politicians reported supporting new pedestrian safety schemes. The majority found the advocacy information surprising, interesting, effectively presented, and could identify suitable local interventions.

Conclusions

This study demonstrates the feasibility of an innovative approach to translational public health by targeting local politicians in a randomised controlled trial. The intervention package was positively viewed and raised interest but changes in interventions were not statistically significance. Longer term supported advocacy may be needed.

Trial Registration

Current Controlled Trials ISRCTN91381117     

Genetic signatures of a range expansion in natura: when clones play leapfrog

The genetic consequences of range expansions have generally been investigated at wide geographical and temporal scales, long after the colonization event. A unique ecological system enabled us to both monitor the colonization dynamics and decipher the genetic footprints of expansion over a very short time period. Each year an epidemic of the poplar rust (Melampsora larici‐populina) expands clonally and linearly along the Durance River, in the Alps. The colonization dynamics observed in 2004 showed two phases with different genetic outcomes. Upstream, fast colonization maintained high genetic diversity. Downstream, the colonization wave progressively faltered, diversity eroded, and differentiation increased, as expected under recurrent founder events. In line with the high dispersal abilities of rust pathogens, we provide evidence for leapfrog dispersal of clones. Our results thus emphasize the importance of colonization dynamics in shaping spatial genetic structure in the face of high gene flow.     

Evaluation of <Emphasis Type="Italic">Galleria mellonella</Emphasis> larvae as an in vivo model for assessing the relative toxicity of food preservative agents

Larvae of Galleria mellonella are widely used for evaluating the virulence of microbial pathogens and for measuring the efficacy of anti-microbial agents and produce results comparable to those that can be obtained using mammals. In this work, the suitability of using G. mellonella larvae to measure the relative toxicity of a variety of food preservatives was evaluated. The response of larvae to eight commonly used food preservatives (potassium nitrate, potassium nitrite, potassium sorbate, sodium benzoate, sodium nitrate, sodium chloride, sodium nitrite and sodium acetate) administered by feeding or by intra-haemocoel injection was measured. A significant correlation between the LD₅₀ (R ²?=?0.8766, p?=?0.0006) and LD₈₀ (R ²?=?0.7629, p?=?0.0046) values obtained due to oral or intra-haemocoel administration of compounds was established. The response of HEp-2 cells to the food preservatives was determined, and a significant correlation (R ²?=?0.7217, p?=?0.0076) between the LD₅₀ values of the compounds administered by feeding in larvae with the IC₅₀ values of the compounds in HEp-2 cells was established. A strong correlation between the LD₅₀ values of the eight food preservatives in G. mellonella larvae and rats (R ²?=?0.6506, p?=?0.0156) was demonstrated. The results presented here indicate that G. mellonella larvae may be used as a model to evaluate the relative toxicity of food preservatives, and the results show a strong positive correlation to those obtained using established cell culture and mammalian models.     

A sharp incisor tool for predator house mice back to the wild

The house mouse (Mus musculus domesticus), as a successful invasive species worldwide, has to forage a variety of resources. Subantarctic mice display among the most notable diet shift from the usual omnivorous–granivorous diet, relying on a larger proportion of terrestrial animal prey. In agreement, a recent study of their mandible morphology evidenced an evolution of their mandible shape to optimize incisor biting and hence seize preys. Here, the incisors themselves are the focus of a morphometric analysis combined with a 3D study of their internal structure, aiming at a comparison between subantarctic populations (Guillou island, Kerguelen archipelago) with a range of western European continental, commensal mice. The predatory foraging behavior of Guillou mice was indeed associated with a sharper bevel of the lower incisor, which appears as an efficient morphology for piercing prey. The incisor of these mice also displays a reduced pulp cavity, suggesting slower eruption counterbalancing a reduced abrasion on such soft food material. The dynamics of the ever‐growing incisor may thus allow adaptive incisor sculpting and participate to the success of mice in foraging diverse resources.     

Evolutionary stasis of a heritable morphological trait in a wild fish population despite apparent directional selection

Comparing observed versus theoretically expected evolutionary responses is important for our understanding of the evolutionary process, and for assessing how species may cope with anthropogenic change. Here, we document directional selection for larger female size in Atlantic salmon, using pedigree‐derived estimates of lifetime reproductive success as a fitness measure. We show the trait is heritable and, thus, capable of responding to selection. The Breeder's Equation, which predicts microevolution as the product of phenotypic selection and heritability, predicted evolution of larger size. This was at odds, however, with the observed lack of either phenotypic or genetic temporal trends in body size, a so‐called “paradox of stasis.” To investigate this paradox, we estimated the additive genetic covariance between trait and fitness, which provides a prediction of evolutionary change according to Robertson's secondary theorem of selection (STS) that is unbiased by missing variables. The STS prediction was consistent with the observed stasis. Decomposition of phenotypic selection gradients into genetic and environmental components revealed a potential upward bias, implying unmeasured factors that covary with trait and fitness. These results showcase the power of pedigreed, wild population studies—which have largely been limited to birds and mammals—to study evolutionary processes on contemporary timescales.     

Transcriptomic analysis of IgG4 Fc-fusion protein degradation in a panel of clonally-derived CHO cell lines using RNASeq

In this study, we report an investigation of a panel of clonally-derived Chinese hamster ovary (CHO) cell lines exhibiting variability in the proportion of full-length IgG4 Fc-fusion protein produced. The recombinant protein was found to be degraded during cell culture into four shorter “clipped” species (three of the four cleavage sites occurred at arginine residues) and preliminary analyses suggested that a host cell enzyme was responsible for proteolysis. To identify the specific enzyme responsible, RNA sequencing was used to identify gene expression differences between the cell lines with a “high” and “low” clipping phenotype. From this analysis, six protease-encoding genes were found to be significantly upregulated in those cell lines yielding the lowest proportion of full-length IgG4 Fc-fusion protein. Four of these protease candidates were deprioritized after examination of their cleavage site specificity. The remaining enzymes, Adam19 and Furin, were found to be capable of cleavage at arginine residues, and inhibitors for both proteases were added to cell-free media to determine if the product degradation could be reduced. While the Adam19 inhibitor had no impact, Furin inhibitor I (specific for the proprotein convertase family of enzymes) was found to result in a 33–39% increase in complete IgG4 Fc-fusion protein when compared with untreated samples.