The Effect of Factor VIII Deficiencies and Replacement and Bypass Therapies on Thrombus Formation under Venous Flow Conditions in Microfluidic and Computational Models

Clinical evidence suggests that individuals with factor VIII (FVIII) deficiency (hemophilia A) are protected against venous thrombosis, but treatment with recombinant proteins can increase their risk for thrombosis. In this study we examined the dynamics of thrombus formation in individuals with hemophilia A and their response to replacement and bypass therapies under venous flow conditions. Fibrin and platelet accumulation were measured in microfluidic flow assays on a TF-rich surface at a shear rate of 100 s⁻¹. Thrombin generation was calculated with a computational spatial-temporal model of thrombus formation. Mild FVIII deficiencies (5–30% normal levels) could support fibrin fiber formation, while severe (<1%) and moderate (1–5%) deficiencies could not. Based on these experimental observations, computational calculations estimate an average thrombin concentration of ∼10 nM is necessary to support fibrin formation under flow. There was no difference in fibrin formation between severe and moderate deficiencies, but platelet aggregate size was significantly larger for moderate deficiencies. Computational calculations estimate that the local thrombin concentration in moderate deficiencies is high enough to induce platelet activation (>1 nM), but too low to support fibrin formation (<10 nM). In the absence of platelets, fibrin formation was not supported even at normal FVIII levels, suggesting platelet adhesion is necessary for fibrin formation. Individuals treated by replacement therapy, recombinant FVIII, showed normalized fibrin formation. Individuals treated with bypass therapy, recombinant FVIIa, had a reduced lag time in fibrin formation, as well as elevated fibrin accumulation compared to healthy controls. Treatment of rFVIIa, but not rFVIII, resulted in significant changes in fibrin dynamics that could lead to a prothrombotic state.     

Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysis

Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for microscopy, enzyme and cell immobilization, and stabilization of protein crystals. Despite of its common use as a crosslinking agent, the mechanism and chemistry involved in glutaraldehyde crosslinking reaction is not yet fully understood. Here we describe feasibility study and results obtained from a new approach to investigate the process of protein crystals stabilization by glutaraldehyde crosslinking. It involves exposure of a model protein crystal (Lysozyme) to glutaraldehyde in alkaline or acidic pH for different incubation periods and reaction arrest by medium exchange with crystallization medium to remove unbound glutaraldehyde. The crystals were subsequently incubated in diluted buffer affecting dissolution of un-crosslinked crystals. Samples from the resulting solution were subjected to protein composition analysis by gel electrophoresis and mass spectroscopy while crosslinked, dissolution resistant crystals were subjected to high resolution X-ray structural analysis. Data from gel electrophoresis indicated that the crosslinking process starts at specific preferable crosslinking site by lysozyme dimer formation, for both acidic and alkaline pH values. These dimer formations were followed by trimer and tetramer formations leading eventually to dissolution resistant crystals. The crosslinking initiation site and the end products obtained from glutaraldehyde crosslinking in both pH ranges resulted from reactions between lysine residues of neighboring protein molecules and the polymeric form of glutaraldehyde. Reaction rate was much faster at alkaline pH. Different reaction end products, indicating different reaction mechanisms, were identified for crosslinking taking place under alkaline or acidic conditions.     

Regulation of the sodium/sulfate co-transporter by farnesoid X receptor alpha

Fxralpha is known to regulate a variety of metabolic processes, including bile acid, cholesterol, and carbohydrate metabolism. In this study, we show direct evidence that Fxralpha is a key player in maintaining sulfate homeostasis. We identified and characterized the sodium/sulfate co-transporter (NaS-1; Slc13a1) as an Fxralpha target gene expressed in the kidney and intestine. Electromobility shift assays, chromatin immunoprecipitation, and promoter reporter studies identified a single functional Fxralpha response element in the second intron of the mouse Slc13a1 gene. Treatment of wild-type mice with GW4064, a synthetic Fxralpha agonist, induced Slc13a1 mRNA in the intestine and kidney. Slc13a1 mRNA was also induced in the kidney and intestine of wild-type, but not Fxralpha-/- mice, after treatment with the hepatotoxin alpha-naphthylisothiocyanate, which is known to result in elevated blood bile acid levels. Finally, we observed a decrease in Slc13a1 mRNA in the kidney and intestine of Fxralpha-/- mice and a corresponding increase in urinary excretion of free sulfates as compared with wild-type mice. These results demonstrate that mouse Slc13a1 is a novel Fxralpha target gene expressed in the kidney and intestine and that in the absence of Fxralpha, mice waste sulfate into the urine. Thus, Fxralpha is necessary for normal sulfate homeostasis in vivo.     

Irreconciliation as practice: resisting impunity and closure in Argentina

In Argentina, irreconciliation is created through everyday practices of vigilance against closure and collective struggles against impunity. In this essay, I show how over several decades since the fall of the dictatorial regime (1976-83), human rights activists and laypeople have devised ways to keep the past alive while attending to injustices through embodied collective engagements with the country's history and its legacies. By examining large protests, the everyday experiences of impunity, and a filmic exploration of kinship bonds and their entanglement with civilian complicity in the repression, the essay illustrates the ways in which irreconciliation is materialized and enacted as a form of social reconstruction many years after state terrorism.     

Using multilayer network analysis to explore the temporal dynamics of collective behavior

Social organisms often show collective behaviors such as group foraging or movement.Collective behaviors can emerge from interactions between group members and may depend on the behavior of key individuals.When social interactions change over time,collective behaviors may change because these behaviors emerge from interactions among individuals.Despite the importance of,and growing interest in,the temporal dynamics of social interactions,it is not clear how to quantify changes in interactions over time or measure their stability.Furthermore,the temporal scale at which we should observe changes in social networks to detect biologically meaningful changes is not always apparent.Here we use multilayer network analysis to quantify temporal dynamics of social networks of the social spider Stegodyphus dumicola and determine how these dynamics relate to individual and group behaviors.We found that social interactions changed over time at a constant rate.Variation in both network structure and the identity of a keystone individual was not related to the mean or variance of the collective prey attack speed.Individuals that maintained a large and stable number of connections,despite changes in network structure,were the boldest individuals in the group.Therefore,social interactions and boldness are linked across time,but group collective behavior is not influenced by the stability of the social network.Our work demonstrates that dynamic social networks can be modeled in a multilayer framework.This approach may reveal biologically important temporal changes to social structure in other systems.     

Controls for Phylogeny and Robust Analysis in Pareto Task Inference

Understanding the tradeoffs faced by organisms is a major goal of evolutionary biology. One of the main approaches for identifying these tradeoffs is Pareto task inference (ParTI). Two recent papers claim that results obtained in ParTI studies are spurious due to phylogenetic dependence (Mikami T, Iwasaki W. 2021. The flipping t-ratio test: phylogenetically informed assessment of the Pareto theory for phenotypic evolution. Methods Ecol Evol. 12(4):696–706) or hypothetical p-hacking and population-structure concerns (Sun M, Zhang J. 2021. Rampant false detection of adaptive phenotypic optimization by ParTI-based Pareto front inference. Mol Biol Evol. 38(4):1653–1664). Here, we show that these claims are baseless. We present a new method to control for phylogenetic dependence, called SibSwap, and show that published ParTI inference is robust to phylogenetic dependence. We show how researchers avoided p-hacking by testing for the robustness of preprocessing choices. We also provide new methods to control for population structure and detail the experimental tests of ParTI in systems ranging from ammonites to cancer gene expression. The methods presented here may help to improve future ParTI studies.     

Insulin-like growth factor binding protein 4 expression parallels luteinizing hormone receptor expression and follicular luteinization in the primate ovary

It has been suggested that locally produced insulin-like growth factor binding protein 4 (IGFBP4) inhibits ovarian follicular growth and ovulation by interfering with IGF action. According to this hypothesis, IGFBP4-expressing follicles should demonstrate atresia, whereas healthy dominant follicles should be devoid of IGFBP4. Alternatively, according to this view, there could be constitutive expression of the inhibitory IGFBP4 but selective expression of an IGFBP4 protease in dominant follicles, allowing the follicle to mature and ovulate because of degradation of the binding protein. To examine these views concerning the role of IGFBP4 in primate follicular selection, we analyzed cellular patterns of IGFs 1 and 2, IGFBP4, and the IGFBP4 protease (pregnancy-associated plasma protein A [PAPP-A]) mRNA expression in ovaries from late follicular phase rhesus monkeys using in situ hybridization. The IGF1 mRNA was not detected, but the IGF2 mRNA was abundant in theca interna and externa of all antral follicles and was present in the granulosa of large preovulatory and ovulatory follicles. The IGFBP4 mRNA was selectively expressed by LH receptor (LHR) mRNA-positive theca interna cells of healthy antral follicles (defined by aromatase and gonadotropin receptor expression) and by LHR-expressing granulosa cells found only in large preovulatory and ovulatory follicles (defined by size and aromatase expression). The PAPP-A mRNA was abundant in granulosa cells of most follicles without obvious relation to IGFBP4 expression. Ovarian IGFBP4 mRNA levels were markedly increased after treatment with the LH analog, hCG, whereas IGF2 and PAPP-A mRNAs were not significantly altered. In summary, IGFBP4 expression appears to be associated with follicular selection, not with atresia, in the monkey ovary. The IGFBP4 is consistently expressed in healthy theca interna and in luteinized granulosa cells, likely under LH regulation. The IGFBP4 protease, PAPP-A, is widely expressed without apparent selectivity for IGFBP4-expressing follicles or for dominant follicles. These observations suggest that IGFBP4 or an IGFBP4 proteolytic product may be involved with LH-induced steroidogenesis and/or luteinization rather than with inhibition of follicular growth.     

Accumulation of heavy metals in epidermal glands of the waterlily (Nymphaeaceae)

This study investigates the anatomical aspects of heavy-metal accumulation in the waterlily (Nymphaea `Aurora', Nymphaeaceae). Epidermal glands were identified by light microscopy on the abaxial side of the leaf laminae and on the epidermis of the rhizome; glandular trichomes were observed in the petiole epidermis. Glands were not observed in the roots. Accumulation of heavy metals in these glands was monitored using a scanning electron microscope equipped for energy-dispersive spectroscopy. Further experiments showed maximal cadmium and calcium accumulation in the mature leaf lamina in daylight, and this accumulation was inhibited by the herbicide 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. These results suggest that, in Nymphaea, heavy metals are accumulated primarily in association with glands found in plant organs that have direct contact with water or mud. Deposition and storage of heavy metals by these glands may represent a stage in the sequestration and detoxification of the metals. Our results raise the possibility of utilizing waterlilies for the removal of heavy metals from polluted environments. Received: 29 April 2000 / Accepted: 8 June 2000