Longitudinal proteomics study of serum changes after allogeneic HSCT reveals potential markers of metabolic complications related to aGvHD.

Even though hematopoietic stem cell transplantation (HSCT) allows successful treatment for many malignant and non-malignant disorders, its curative potential remains limited by severe side effects, including infections and other transplant-related complications such as graft-versus-host disease (GvHD). This study examined changes in serum proteome via high-performance two-dimensional gel electrophoresis (2-DE) during HSCT to search for diagnostic biomarkers for post-HSCT complications. Longitudinal proteomic analysis revealed proteins related to metabolic complications and hemolytic anemia. Retinol-binding protein 4 (RBP4), a reliable marker of insulin resistance, was identified, and is possibly associated with the onset mechanism of acute graft-versus-host disease (aGvHD) and/or skin GvHD. Although the cause of insulin resistance is not fully understood, it is thought to be associated with adipocytes inflammation induced by RBP4, iron overload and hemolytic anemia after HSCT, as observed in this study. The present study has demonstrated that insulin resistance and metabolic complications could be immediate complications after transplantation and are associated with aGvHD. The biomarkers revealed in this study are promising tools to be used for improving the early diagnosis of HSCT-associated complications, especially aGvHD, possibly even before clinical manifestations.

Detailed Analysis of Zebrafish Larval Behaviour in the Light Dark Challenge Assay Shows That Diel Hatching Time Determines Individual Variation.

Research on stress coping style, i.e., the response of an organism to adverse conditions, which is constant over time and context, gained momentum in recent years, to better understand behavioural patterns in animal welfare. However, knowledge about the ontogeny of stress coping style is still limited. Here, we performed a detailed analysis of the light dark challenge behavioural assay in zebrafish larvae, where after acclimation in ambient light sudden alternating dark and light phases elicit an anxiety-like response. A principal component analysis on parameters related to locomotion (distance moved, swimming velocity, acceleration, mobility) and directionality (angular velocity, meandering of swimming path) revealed independence between the parameters determined in the light and the dark phases of the assay, indicating unrelated generalised behaviours per phase. However, high collinearity was observed between behavioural parameters within the same phase, indicating a robust response to the stimulus within behavioural phenotypes. Subsequently, this assay was used to determine the correlation between individual hatching time and the behavioural phenotype. The results show that fish that had hatched during daytime have a stronger behavioural response to the dark phase at 5 days post-fertilisation in locomotion related parameters and a weaker response in directionality related parameters, than fish that had hatched during nighttime. These results show that behavioural responses to the light dark challenge assay are robust and can be generalised for the light and the dark phase, and that diel hatching time may determine the behavioural phenotype of an individual.

Stabilin 1 and 2 are important regulators for cellular uptake of apolipoprotein B-containing lipoproteins in zebrafish.

BACKGROUND AND AIMS: Scavenger receptors form a superfamily of membrane-bound receptors that bind and internalize different types of ligands, including pro-atherogenic oxidized low-density lipoproteins (oxLDLs). In vitro studies have indicated a role for the liver sinusoidal endothelial cell receptors stabilin 1 (stab1) and 2 (stab2) in oxLDL clearance. In this study, we evaluated the potential role of stab1 and stab2 in lipoprotein uptake in zebrafish, an upcoming model for studying cholesterol metabolism and atherosclerosis. METHODS: Lipoproteins were injected in the duct of Cuvier of wild-type (ABTL) or stab1 and stab2 mutant (stab1-/-stab2-/-) zebrafish larvae at 3 days post-fertilization. To examine the effect of stabilin deficiency on lipoprotein and cholesterol metabolism, zebrafish larvae were challenged with a high cholesterol diet (HCD; 4% w/w) for 10 days. RESULTS: Lipoprotein injections showed impaired uptake of both LDL and oxLDL into the vessel wall of caudal veins of stab1-/-stab2-/- zebrafish, which was paralleled by redistribution to tissue macrophages. Total body cholesterol levels did not differ between HCD-fed stab1-/-stab2-/- and ABTL zebrafish. However, stab1-/-stab2-/- larvae exhibited 1.4-fold higher mRNA expression levels of ldlra involved in (modified) LDL uptake, whereas the expression levels of scavenger receptors scarb1 and cd36 were significantly decreased. CONCLUSIONS: We have shown that stabilins 1 and 2 have an important scavenging function for apolipoprotein B-containing lipoproteins in zebrafish and that combined deficiency of these two proteins strongly upregulates the clearance of lipoproteins by macrophages within the caudal vein. Our current study highlights the use of zebrafish as model to study lipoprotein metabolism and liver sinusoidal endothelial cell function.

Lrp5 Mutant and Crispant Zebrafish Faithfully Model Human Osteoporosis, Establishing the Zebrafish as a Platform for CRISPR-Based Functional Screening of Osteoporosis Candidate Genes.

Genomewide association studies (GWAS) have improved our understanding of the genetic architecture of common complex diseases such as osteoporosis. Nevertheless, to attribute functional skeletal contributions of candidate genes to osteoporosis-related traits, there is a need for efficient and cost-effective in vivo functional testing. This can be achieved through CRISPR-based reverse genetic screens, where phenotyping is traditionally performed in stable germline knockout (KO) mutants. Recently it was shown that first-generation (F0) mosaic mutant zebrafish (so-called crispants) recapitulate the phenotype of germline KOs. To demonstrate feasibility of functional validation of osteoporosis candidate genes through crispant screening, we compared a crispant to a stable KO zebrafish model for the lrp5 gene. In humans, recessive loss-of-function mutations in LRP5, a co-receptor in the Wnt signaling pathway, cause osteoporosis-pseudoglioma syndrome. In addition, several GWAS studies identified LRP5 as a major risk locus for osteoporosis-related phenotypes. In this study, we showed that early stage lrp5 KO larvae display decreased notochord mineralization and malformations of the head cartilage. Quantitative micro-computed tomography (micro-CT) scanning and mass-spectrometry element analysis of the adult skeleton revealed decreased vertebral bone volume and bone mineralization, hallmark features of osteoporosis. Furthermore, regenerating fin tissue displayed reduced Wnt signaling activity in lrp5 KO adults. We next compared lrp5 mutants with crispants. Next-generation sequencing analysis of adult crispant tissue revealed a mean out-of-frame mutation rate of 76%, resulting in strongly reduced levels of Lrp5 protein. These crispants generally showed a milder but nonetheless highly comparable skeletal phenotype and a similarly reduced Wnt pathway response compared with lrp5 KO mutants. In conclusion, we show through faithful modeling of LRP5-related primary osteoporosis that crispant screening in zebrafish is a promising approach for rapid functional screening of osteoporosis candidate genes. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Multiple biomarkers of sepsis identified by novel time-lapse proteomics of patient serum.

Serum components of sepsis patients vary with the severity of infection, the resulting inflammatory response, per individual, and even over time. Tracking these changes is crucial in properly treating sepsis. Hence, several blood-derived biomarkers have been studied for their potential in assessing sepsis severity. However, the classical approach of selecting individual biomarkers is problematic in terms of accuracy and efficiency. We therefore present a novel approach for detecting biomarkers using longitudinal proteomics data. This does not require a predetermined set of proteins and can therefore reveal previously unknown related proteins. Our approach involves examining changes over time of both protein abundance and post-translational modifications in serum, using two-dimensional gel electrophoresis (2D-PAGE). 2D-PAGE was conducted using serum from n = 20 patients, collected at five time points, starting from the onset of sepsis. Changes in protein spots were examined using 49 spots for which the signal intensity changed by at least two-fold over time. These were then screened for significant spikes or dips in intensity that occurred exclusively in patients with adverse outcome. Individual level variation was handled by a mixed effects model. Finally, for each time transition, partial correlations between spots were estimated through a Gaussian graphical model (GGM) based on the ridge penalty. Identifications of spots of interest by tandem mass spectrometry revealed that many were either known biomarkers for inflammation (complement components), or had previously been suggested as biomarkers for kidney failure (haptoglobin) or liver failure (ceruloplasmin). The latter two are common complications in severe sepsis. In the GGM, many of the tightly connected spots shared known biological functions or even belonged to the same protein; including hemoglobin chains and acute phase proteins. Altogether, these results suggest that our screening method can successfully identify biomarkers for disease states and cluster biologically related proteins using longitudinal proteomics data derived from 2D-PAGE.

Gender Stereotypes in Science Education Resources: A Visual Content Analysis.

More men are studying and working in science fields than women. This could be an effect of the prevalence of gender stereotypes (e.g., science is for men, not for women). Aside from the media and people's social lives, such stereotypes can also occur in education. Ways in which stereotypes are visible in education include the use of gender-biased visuals, language, teaching methods, and teachers' attitudes. The goal of this study was to determine whether science education resources for primary school contained gender-biased visuals. Specifically, the total number of men and women depicted, and the profession and activity of each person in the visuals were noted. The analysis showed that there were more men than women depicted with a science profession and that more women than men were depicted as teachers. This study shows that there is a stereotypical representation of men and women in online science education resources, highlighting the changes needed to create a balanced representation of men and women. Even if the stereotypical representation of men and women in science is a true reflection of the gender distribution in science, we should aim for a more balanced representation. Such a balance is an essential first step towards showing children that both men and women can do science, which will contribute to more gender-balanced science and technology fields.