Search

•Carnitine pools in stored human and murine RBCs are regulated by genetic polymorphisms in the SLC22A16 and SLC22A5 transporters.•Carnitine pools fuel the Lands cycle for damaged membrane lipid repair during RBC aging, affecting hemolysis in vitro and in vivo.

Permanent availability of red blood cells (RBCs) for transfusion depends on refrigerated storage, during which morphologically altered RBCs accumulate. Among these, a subpopulation of small RBCs, comprising type III echinocytes, spheroechinocytes, and spherocytes and defined as storage-induced microerythrocytes (SMEs), could be rapidly cleared from circulation posttransfusion. We quantified the proportion of SMEs in RBC concentrates from healthy human volunteers and assessed correlation with transfusion recovery, investigated the fate of SMEs upon perfusion through human spleen ex vivo, and explored where and how SMEs are cleared in a mouse model of blood storage and transfusion. In healthy human volunteers, high proportion of SMEs in long-stored RBC concentrates correlated with poor transfusion recovery. When perfused through human spleen, 15% and 61% of long-stored RBCs and SMEs were cleared in 70 minutes, respectively. High initial proportion of SMEs also correlated with high retention of RBCs by perfused human spleen. In the mouse model, SMEs accumulated during storage. Transfusion of long-stored RBCs resulted in reduced posttransfusion recovery, mostly due to SME clearance. After transfusion in mice, long-stored RBCs accumulated predominantly in spleen and were ingested mainly by splenic and hepatic macrophages. In macrophage-depleted mice, splenic accumulation and SME clearance were delayed, and transfusion recovery was improved. In healthy hosts, SMEs were cleared predominantly by macrophages in spleen and liver. When this well-demarcated subpopulation of altered RBCs was abundant in RBC concentrates, transfusion recovery was diminished. SME quantification has the potential to improve blood product quality assessment. This trial was registered at www.clinicaltrials.gov as #NCT02889133.

Permanent availability of red blood cells (RBCs) for transfusion depends on refrigerated storage, during which morphologically altered RBCs accumulate. Among these, a subpopulation of small RBCs, comprising type III echinocytes, spheroechinocytes, and spherocytes and defined as storage-induced microerythrocytes (SMEs), could be rapidly cleared from circulation posttransfusion. We quantified the proportion of SMEs in RBC concentrates from healthy human volunteers and assessed correlation with transfusion recovery, investigated the fate of SMEs upon perfusion through human spleen ex vivo, and explored where and how SMEs are cleared in a mouse model of blood storage and transfusion. In healthy human volunteers, high proportion of SMEs in long-stored RBC concentrates correlated with poor transfusion recovery. When perfused through human spleen, 15% and 61% of long-stored RBCs and SMEs were cleared in 70 minutes, respectively. High initial proportion of SMEs also correlated with high retention of RBCs by perfused human spleen. In the mouse model, SMEs accumulated during storage. Transfusion of long-stored RBCs resulted in reduced posttransfusion recovery, mostly due to SME clearance. After transfusion in mice, long-stored RBCs accumulated predominantly in spleen and were ingested mainly by splenic and hepatic macrophages. In macrophage-depleted mice, splenic accumulation and SME clearance were delayed, and transfusion recovery was improved. In healthy hosts, SMEs were cleared predominantly by macrophages in spleen and liver. When this well-demarcated subpopulation of altered RBCs was abundant in RBC concentrates, transfusion recovery was diminished. SME quantification has the potential to improve blood product quality assessment. This trial was registered at www.clinicaltrials.govas #NCT02889133.

Large scale genomics studies and vein-to-vein databases have started to reveal that donor biology and genetics influence red blood cell (RBC) storability and transfusion outcomes. To further delve into this concept, here we performed metabolomics analyses of 13,091 packed RBC units from donors enrolled in the Recipient Epidemiology and Donor Evaluation (REDS) RBC Omics study. End of storage (day 42) units were tested for metabolomics and hemolytic propensity. Donors ranking in the 5 thand 95 thpercentile were contacted again and invited to donate a second unit of blood, which was stored again for 42 days (n=643). Correlation of end of storage metabolomics measurements for the first (index) and second (recalled) donation identified a core of metabolites involved in carnitine synthesis and acyl-carnitine metabolism as the most reproducible within the same donor across multiple donations ( Figure 1.A). Carnitine and its precursors, methyl- and trimethyl-lysine were the most significantly reproducible of all the metabolites tested in this study ( Figure 1.A). Association of L-carnitine measurements to genomics data (i.e., 879,000 single nucleotide polymorphisms that were assayed via a precision transfusion medicine array developed for this study - Figure 1.B) identified non-synonymous coding polymorphisms in the carnitine transporter SLC22A16 as a critical genetic factor contributing to inter-donor heterogeneity in end of storage carnitine levels ( Figure 1.B). Donors carrying two alleles of this SNP were characterized by the lowest L-carnitine levels, and associated depletion of the whole carnitine pool. Functionally, stored RBCs with the lowest levels of carnitine pools were characterized by significant elevation in in vitrohemolysis and the highest degree of vesiculation, in parallel to increases in lipid peroxidation markers (hydroxy-eicosatetraenoic - HETEs and hydroxy-octadecadienoic acids - HODEs).

Serotonin (5-hydroxytryptamine, 5-HT) is a well-known neurotransmitter that is involved in a growing number of functions in peripheral tissues. Recent studies have shown nonpharmacological functions of 5-HT linked to its chemical properties. Indeed, it was reported that 5-HT may, on the one hand, bind lipid membranes and, on the other hand, protect red blood cells through a mechanism independent of its specific receptors. To better understand these underevaluated properties of 5-HT, we combined biochemical, biophysical, and molecular dynamics simulations approaches to characterize, at the molecular level, the antioxidant capacity of 5-HT and its interaction with lipid membranes. To do so, 5-HT was added to red blood cells and lipid membranes bearing different degrees of unsaturation. Our results demonstrate that 5-HT acts as a potent antioxidant and binds with a superior affinity to lipids with unsaturation on both alkyl chains. We show that 5-HT locates at the hydrophobic-hydrophilic interface, below the glycerol group. This interfacial location is stabilized by hydrogen bonds between the 5-HT hydroxyl group and lipid headgroups and allows 5-HT to intercept reactive oxygen species, preventing membrane oxidation. Experimental and molecular dynamics simulations using membrane enriched with oxidized lipids converge to further reveal that 5-HT contributes to the termination of lipid peroxidation by direct interaction with active groups of these lipids and could also contribute to limit the production of new radicals. Taken together, our results identify 5-HT as a potent inhibitor of lipid peroxidation and offer a different perspective on the role of this pleiotropic molecule.

Cytokine release reactions (CRR) induced by platinum-based chemotherapy, manifesting with fever, chills and rigors, are poorly understood and not easily prevented by usual premedication or desensitization.

The last decade has seen a considerable growth in data generated by scientific instrumentation. Accountability requirements have grown similarly, requiring forensic toxicology laboratories to generate summary of findings to funders, stakeholders and the general public. This work aims to show how the programming language R and its integrated development environment (IDE) RStudio can be harnessed to speed up data analysis and produce summary reports in three different forensic toxicology applications.

ABSTRACTPlasmodium falciparumis transmitted from humans to Anophelesmosquito vectors via the sexual erythrocytic forms termed gametocytes. Erythrocyte filtration through microsphere layers (microsphiltration) had shown that circulating gametocytes are deformable. Compounds reducing gametocyte deformability would induce their splenic clearance, thus removing them from the blood circulation and blocking malaria transmission. The hand-made, single-sample prototype for microsphiltration was miniaturized to a 96-well microtiter plate format, and gametocyte retention in the microsphere filters was quantified by high-content imaging. The stiffening activity of 40 pharmacological compounds was assessed in microtiter plates, using a small molecule (calyculin) as a positive control. The stiffening activity of calyculin was assessed in spleen-mimetic microfluidic chips and in macrophage-depleted mice. Marked mechanical retention (80% to 90%) of mature gametocytes was obtained in microplates following exposure to calyculin at concentrations with no effect on parasite viability. Of the 40 compounds tested, including 20 antimalarials, only 5 endoperoxides significantly increased gametocyte retention (1.5- to 2.5-fold; 24 h of exposure at 1 μM). Mature gametocytes exposed to calyculin accumulated in microfluidic chips and were cleared from the circulation of macrophage-depleted mice as rapidly as heat-stiffened erythrocytes, thus confirming results obtained using the microsphiltration assay. An automated miniaturized approach to select compounds for their gametocyte-stiffening effect has been established. Stiffening induces gametocyte clearance both in vitroand in vivo. Based on physiologically validated tools, this screening cascade can identify novel compounds and uncover new targets to block malaria transmission. Innovative applications in hematology are also envisioned.

This study provided validity evidence for the Godin-Shephard Leisure-Time Physical Activity Questionnaire (GSLTPAQ) to classify respondents into activeand insufficiently activecategories. Members of a fitness center [45 women and 55 men; mean (SD) age = 45.5 (10.6) yr.] completed the questionnaire. Using only moderate and strenuous scores, those with a leisure score index ≥ 24 were classified as active; those with a score ≤ 23 were classified as insufficiently active. VO2max, percentage of body fat, and electronic records of fitness center attendance were the validation variables. In a visit to the fitness center, participants completed the GSLTPAQ and a certified exercise specialist performed a physical fitness evaluation. A multivariate analysis of covariance (MANCOVA) indicated the group of respondents classified as activehad higher VO2max and lower percentage of body fat than the group of respondents classified as insufficiently active. An analysis of covariance (ANCOVA) indicated the group of respondents classified as active had higher electronic records of fitness center attendance than the group of respondents classified as insufficiently active. Therefore, these pieces of validity evidence support the use of the questionnaire's classification system among healthy adults.

Lu, Cheng, and Chen (2013) faced one of the most common challenges encountered in longitudinal studies: follow-up attrition. Using a correlational prospective design, 464 volunteers completed a questionnaire that measured the constructs of the theory of planned behavior, and subsequently 154 of them provided physical activity data at a 6-month follow-up. The proportion of participants (66.8%) for whom the investigators were not able to gather information on the behavioral outcome at follow-up may reflect a form of selection bias that may affect both the validity and generalizability of study results. Lu, et al.'s (2013) study is used here to explore the implication of follow-up attrition on the results and inference, to review what information should be reported in a scientific paper in such situations, and to give practical tips to handle follow-up attrition.

The aim of this study was to assess the effects of implementation intentions on leisure-time physical activity, taking into account the stability of intention. At baseline (T0), 349 participants completed a psychosocial questionnaire and were randomly assigned to implementation intention or control condition. Three months after baseline assessment (T1), participants in the experimental group were asked to plan where, when, and how they would exercise. Leisure-time physical activity was assessed 3 mo. later (i.e., at 6-mo. follow-up; T2). The intervention had no significant effect on physical activity at 6-mo. follow-up. However, a significant interaction of group and intention stability was observed, with the effect of the intervention on behaviour statistically significant only among those with unstable intention. Intention stability thus moderated the effect of the intervention, i.e., the intervention was more successful among individuals who needed support to change (unstable intenders).

cAMP and intracellular Ca2+ are important second messengers involved in mammalian follicular growth and oocyte meiotic maturation. We investigated the capacity of the neurohormone serotonin (5-hydroxytryptamine, 5-HT) to regulate intracellular cAMP and Ca2+ in mouse oocytes and surrounding cumulus cells. On the basis of a reverse transcription-polymerase chain reaction study, 5-HT7 receptor mRNA is expressed in cumulus cells, oocytes, and embryos up to the four-cell stage, and 5-HT2A and 5-HT2B receptor mRNAs are expressed in cumulus cells only, whereas 5-HT2C, 5-HT4, and 5-HT6 receptors are expressed in neither oocytes nor cumulus cells. The addition of 5-HT (10 nM to 10 µM) to isolated metaphase II oocytes had no effect on their internal cAMP or Ca2+ levels, whereas it caused dose-dependent cAMP and Ca2+ increases in cumulus cells. This cAMP increase in cumulus cells could be mimicked by 5-HT agonists with the following order of potency: 5-HT > 8-hydroxy-2-(di-n-propylamino) tetralin = α-methyl-5-HT = 5-carboxamidotryptamine maleate > 2-[1-(4-piperonyl)piperazinyl]benzo-triazole, thereby supporting a preferential involvement of 5-HT7 receptors. As measured with cumulus cells preloaded with fura-2/acetoxymethyl ester (AM), the addition of 5-HT also caused dose-dependent Ca2+ increases, which were probably linked to detected 5-HT2A and 5-HT2B receptors. Adding the Ca2+ ionophore ionomycin to cumulus cells resulted in both Ca2+ and cAMP elevations, whereas preincubation of cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM abolished the 5-HT-induced Ca2+ increase and reduced the cAMP increase, indicating cross-talk between the 5-HT-sensitive Ca2+ and cAMP pathways. Our results show that 5-HT may be a local regulator in mouse cumulus-oocyte complexes through its actions on cAMP and Ca2+ signaling, as mediated by 5-HT2A, 5-HT2B, and 5-HT7 receptors.

Serotonin (5-hydroxytryptamine [5-HT]), is a neurohormone found in various nonneural tissues, including the gonads of many invertebrates, in which it regulates spawning and oocyte meiotic maturation. The possibility that a local serotonergic network might also exist in the female gonads of vertebrate species, including mammals, remains poorly documented. To clarify this possibility, we investigated mouse cumulus cells, oocytes, and embryos for three key serotonergic components, namely, 5-HT itself; the rate-limiting enzyme for its production, tryptophan hydroxylase 1 (TPH1); and the 5-HT-specific transporter (SLC6A4) required for modulating its cellular effects. Using a combination of reverse transcription-polymerase chain reaction analysis and immunofluorescence confocal microscopy, we showed that mouse cumulus cells, oocytes, and embryos contain 5-HT and SLC6A4, while only cumulus cells possess the 5-HT-producing enzyme TPH1 and may thus be the local source of 5-HT observed in their neighboring cells. With a semiquantitative assay in single cells, we demonstrated that 5-HT can actively be taken up by isolated oocytes when it is supplied exogenously in vitro. This 5-HT transport in isolated oocytes is driven by a classical serotonin transporter, expressed up to the blastocyst stage, that is sensitive to the antidepressants fluoxetine and fluvoxamine, which belong to the selective serotonin reuptake inhibitor family. All together, our results show that 5-HT may be produced locally by cumulus cells and that it can be actively taken up by mammalian oocytes and embryos as part of a likely larger serotonergic network possibly regulating various developmental processes much earlier than previously thought.

Artificial substrata are increasingly used to study periphyton, but their ability to reproduce natural substrata remains controversial. Although many studies have made contemporaneous comparisons of periphyton assemblages on artificial and natural substrata at one or a few sites, no broadly based comparison exists. We therefore surveyed the literature to establish conditions under which artificial substrata satisfactorily mimic both the quantity and the quality of natural periphyton assemblages. In general, epilithon was underestimated by the artificial substrata; epiphyton was overestimated, but less severely. These trends were significantly affected by the time available for colonization of the artificial substrata before sampling, site trophy, ambient temperature, and whether the study was conducted in a lake or in running water. Neither the composition of the substratum nor its orientation appeared important. Natural diatom assemblages were usually well simulated by those on artificial substrata, whereas both epilithic and epiphytic green and bluegreen algae were severely underrepresented on the artificial substrata. Since artificial substrata often misrepresent both the quantity and the quality of natural periphyton, they should be used with more caution, especially in intersite and interseason surveys.

Purpose

Roussel: Zimmer Biomet: Research Funding. MARIN:Zimmer Biomet: Research Funding. Spitalnik:Hemanext: Membership on an entity's Board of Directors or advisory committees; Tioma, Inc.: Consultancy. Hermine:AB science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Research Funding. Buffet:Zimmer Biomet: Research Funding. Amireault:Zimmer Biomet: Research Funding.

Buffet: Zimmer Biomet: Research Funding. Hermine:Celgene: Research Funding; Novartis: Research Funding; AB science: Consultancy, Equity Ownership, Honoraria, Research Funding. Amireault:Zimmer Biomet: Research Funding.

Background

No relevant conflicts of interest to declare.

Introduction: Storage of red blood cells (RBCs) for transfusion purposes is accompanied by a number of morphological and biochemical changes (storage lesions) that reduce post-transfusion survival/efficacy and increase risk for adverse reactions in the recipients. The clearance of altered and older RBCs from circulation is triggered by the clustering of Band 3, an aggregate state that is recognized by a low-affinity naturally occurring IgG antibody (Nab). Considering the key role of Band 3 in the maintenance of RBC structure and survival, elucidation of functional and structural modifications of Band 3 during storage should lead to new approaches aiming to improve RBC storage and post-transfusion viability.

No relevant conflicts of interest to declare.

The storage lesion encompasses a series of biochemical and molecular modifications that alter erythrocytes during hypothermic storage, reducing transfusion yield. Indeed, in humans, up to 25% of transfused erythrocytes are cleared from the circulation in a few hours (Luten et al., 2008). The mechanisms underlying this rapid clearance are not fully elucidated but it is reasonable to assume that these erythrocyte alterations are sensed by the spleen, resulting in retention. Among those, membrane shedding may have a major impact on post-transfusion clearance of erythrocytes since it causes a progressive decrease in the surface-volume ratio of the cell, leading to the loss of its flexible biconcave shape. The proportion of "damaged" erythrocytes cleared in the hours following transfusion increases with the duration of storage while the deformability measured by ektacytometry (Frank et al., 2013) progressively decreases during this period. To characterize the morphological alteration of stored erythrocytes, we used imaging flow cytometry (Imagestream X Mark II, AMNIS°). This technology allows a simultaneous high-speed multispectral imaging of cells in brightfield, darkfield, and 9 channels of fluorescence. It combines the ability of conventional flow cytometry to analyze a very high number of events with a powerful exploration of cell morphology.