Effect of mechanical fatigue on commercial bioprosthetic TAVR valve mechanical and microstructural properties.

Valvular structural deterioration is of particular concern for transcatheter aortic valve replacements due to their suspected shorter longevity and increasing use in younger patient populations. In this work we investigated the mechanical and microstructural changes in commercial TAVR valves composed of both glutaraldehyde fixed bovine and porcine pericardium (GLBP and GLPP) following accelerated wear testing (AWT) as outlined in ISO 5840 standards. This provided greater physiological relevance to the loading compared to previous studies and by utilizing digital image correlation we were able to obtain strain contours for each leaflet pre and post fatigue and identify sites of fatigue damage. The areas of greatest change in mechanical strain for each leaflet were then further probed using biaxial tensile testing, confocal microscopy, and electron microscopy. It was observed that overall strain decreased in the GLPP valves following AWT of 200 million cycles while the GLBP valve showed an increase in overall strain. Biaxial tensile testing showed a statistically significant reduction in stress for GLPP while no significant changes were seen for GLBP. Both confocal and electron microscopy showed a disruption to the gross collagen organization and fibrillar structure, including fragmentation, for GLPP but only the former for GLBP. However, further test data is required to confirm these findings and to provide a better understanding of this fatigue pathway is required such that it can be incorporated into both valve design and selection processes to improve overall longevity for both GLPP and GLBP devices.

Boundary Frustration in Double-Gyroid Thin Films.

Self-consistent field theory for thin films of AB diblock polymers in the double-gyroid phase reveals that in the absence of preferential wetting of monomer species at the film boundaries, films with the (211) plane oriented parallel to the boundaries are more stable than other orientations, consistent with experimental results. This preferred orientation is explained in the context of boundary frustration. Specifically, the angle of intersection between the A/B interface and the film boundary, the wetting angle, is thermodynamically restricted to a narrow range of values. Most termination planes in the double gyroid cannot accommodate this narrow range of wetting angles without significant local distortion relative to the bulk morphology; the (211)-oriented termination plane with the "double-wave" pattern produces relatively minimal distortion, making it the least frustrated boundary. The principle of boundary frustration provides a framework to understand the relative stability of termination planes for complex ordered block polymer phases confined between flat, nonpreferential boundaries.

spinDrop: a droplet microfluidic platform to maximise single-cell sequencing information content.

Droplet microfluidic methods have massively increased the throughput of single-cell sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples and the overall RNA capture efficiency is lower. These drawbacks stem from the lack of strategies to enrich for high-quality material or specific cell types at the moment of cell encapsulation and the absence of implementable multi-step enzymatic processes that increase capture. Here we alleviate both bottlenecks using fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei, fixed cells or target cell types and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half. We harness these properties to deliver a high-quality molecular atlas of mouse brain development, despite starting with highly damaged input material, and provide an atlas of nascent RNA transcription during mouse organogenesis. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.

Ribosome profiling in the Symbiodiniacean dinoflagellate Fugacium kawagutii shows coordinated protein synthesis of enzymes in different pathways at different times of day.

Dinoflagellates respond to daily changes in light and dark by changes in cellular metabolism, yet the mechanisms used are still unclear. For example, Fugacium (previously Symbiodinium) kawagutii shows little difference in the transcriptome between day and night suggesting little transcriptional control over gene expression. Here, we have performed ribosome profiling at 2 h intervals over a daily light-dark cycle to assess the degree to which protein synthesis rates might change over the daily cycle. The number of F. kawagutii coding sequences with significant differences in the number of ribosome-protected fragments (RPF) over the 24-h cycle was 2923 using JTK_Cycle and 3655 using ECHO. The majority of the regulated transcripts showed peak translation at the onset of the dark period. The regulated sequences were assigned to different KEGG pathways and transcripts that were translated at roughly the same time were termed concurrently regulated. Both analyses revealed concurrent regulation of many transcripts whose gene products were involved in spliceosome or lysosome biogenesis with peak translation rates around the onset of the dark period, while others, involved in nitrate metabolism and ribosomal proteins, were preferentially translated around the onset of the day phase or the end of the night phase, respectively. In addition, some sequences involved in DNA synthesis were preferentially translated at the end of the day. We conclude that light-dark cycles seem able to synchronize translation of some transcripts encoding proteins involved in a range of different cellular processes, and propose that these changes may help the cells adapt and alter their metabolism as a function of the time of day.

Positional influence on cellular transcriptional identity revealed through spatially segmented single-cell transcriptomics.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for describing cell states. Identifying the spatial arrangement of these states in tissues remains challenging, with the existing methods requiring niche methodologies and expertise. Here, we describe segmentation by exogenous perfusion (SEEP), a rapid and integrated method to link surface proximity and environment accessibility to transcriptional identity within three-dimensional (3D) disease models. The method utilizes the steady-state diffusion kinetics of a fluorescent dye to establish a gradient along the radial axis of disease models. Classification of sample layers based on dye accessibility enables dissociated and sorted cells to be characterized by transcriptomic and regional identities. Using SEEP, we analyze spheroid, organoid, and in vivo tumor models of high-grade serous ovarian cancer (HGSOC). The results validate long-standing beliefs about the relationship between cell state and position while revealing new concepts regarding how spatially unique microenvironments influence the identity of individual cells within tumors.

Insights into daily metabolic changes of the dinoflagellate Lingulodinium from ribosome profiling.

The dinoflagellate Lingulodinium specializes its metabolism to perform different tasks better at specific times of day. For example, cells are specialized for photosynthesis during the day and bioluminescence and cell division at night. These rhythms are circadian as they are controlled by an endogenous circadian clock whose mechanism is currently unknown. Despite this, the metabolic rhythms follow coordinated changes in gene expression that occur at a translational level. These changes are revealed by ribosome profiling, a surrogate measure of protein synthesis rates in vivo. Lingulodinium regulates the synthesis rate of over three thousand transcripts. Peak synthesis rates for the different transcripts are clustered around three different times over a light/dark cycle. Furthermore, transcripts involved in the same metabolic process are coordinately regulated. We review the basic principles underlying the correlation of coordinated translation of cell metabolic pathway enzymes with known circadian rhythms, and offer examples where previously unsuspected rhythms are suggested by synchronized changes in gene expression.

Diffusion of surfactant from a micellar solution to a bare interface. 1. Absorbing boundary.

We analyze dynamic adsorption of surfactant from a micellar solution to a rapidly created surface that acts as an absorbing boundary for surfactant monomers (single molecules), along which the monomer concentration vanishes, with no direct micelle adsorption. This somewhat idealized situation is analyzed as a prototype for situations in which strong suppression of monomer concentration accelerates micelle dissociation, and will be used as a starting point for analysis of more realistic boundary conditions in subsequent work. We present scaling arguments and approximate models for particular time and parameter regimes and compare the resulting predictions to numerical simulations of the reaction-diffusion equations for a polydisperse system containing surfactant monomers and clusters of arbitrary aggregation number. The model considered here exhibits an initial period of rapid shrinkage and ultimate dissociation of micelles within a narrow region near the interface. This opens a micelle-free region near the interface after some time τe, the width of which increases as t1/2 at times t≫τe. In systems that exhibit disparate fast and slow bulk relaxation times τ1 and τ2 in response to small perturbations, τe is usually comparable to or greater than τ1 but much less than τ2. Such systems exhibit a wide intermediate time regime τe

Orchestrated translation specializes dinoflagellate metabolism three times per day.

Many cells specialize for different metabolic tasks at different times over their normal ZT cycle by changes in gene expression. However, in most cases, circadian gene expression has been assessed at the mRNA accumulation level, which may not faithfully reflect protein synthesis rates. Here, we use ribosome profiling in the dinoflagellate Lingulodinium polyedra to identify thousands of transcripts showing coordinated translation. All of the components in carbon fixation are concurrently regulated at ZT0, predicting the known rhythm of carbon fixation, and many enzymes involved in DNA replication are concurrently regulated at ZT12, also predicting the known rhythm in this process. Most of the enzymes in glycolysis and the TCA cycle are also regulated together, suggesting rhythms in these processes as well. Surprisingly, a third cluster of transcripts show peak translation at approximately ZT16, and these transcripts encode enzymes involved in transcription, translation, and amino acid biosynthesis. The latter has physiological consequences, as measured free amino acid levels increase at night and thus represent a previously undocumented rhythm in this model. Our results suggest that ribosome profiling may be a more accurate predictor of changed metabolic state than transcriptomics.

Radiopharmaceutical Quality Control Considerations for Accelerator-Produced Actinium Therapies.

Background: Alpha-particle-emitting radiotherapies are of great interest for the treatment of disseminated cancer. Actinium-225 (225Ac) produces four α-particles through its decay and is among the most attractive radionuclides for use in targeted radiotherapy applications. However, supply issues for this isotope have limited availability and increased cost for research and translation. Efforts have focused on accelerator-based methods that produce 225Ac in addition to long-lived 227Ac. Objective: The authors investigated the impact of 225Ac/227Ac material in the radiolabeling and radiopharmaceutical quality control evaluation of a DOTA chelate-conjugated peptide under good manufacturing practices. The authors use an automated module under identical conditions with either generator or accelerator-produced actinium radiolabeling. Methods: The authors have performed characterization of the radiolabeled products, including thin-layer chromatography, high-pressure liquid chromatography, gamma counting, and high-energy resolution gamma spectroscopy. Results: Peptide was radiolabeled and assessed at >95% radiochemical purity with high yields for generator produced 225Ac. The radiolabeling results produced material with subtle but detectable differences when using 225Ac/227Ac. Gamma spectroscopy was able to identify peptide initially labeled with 227Th, and at 100 d for quantification of 225Ac-bearing peptide. Conclusion: Peptides produced using 225Ac/227Ac material may be suitable for translation, but raise new issues that include processing times, logistics, and contaminant detection.

An overview of transcription in dinoflagellates.

Dinoflagellates are a vital diverse family of unicellular algae widespread in various aquatic environments. Typically large genomes and permanently condensed chromosomes without histones make these organisms unique among eukaryotes in terms of chromatin structure and gene expression. Genomic and transcriptomic sequencing projects have provided new insight into the genetic foundation of dinoflagellate behaviors. Genes in tandem arrays, trans-splicing of mRNAs and lower levels of transcriptional regulation compared to other eukaryotes all contribute to the differences seen. Here we present a general overview of transcription in dinoflagellates based on previously described work.

Nonlinear dynamics in micellar surfactant solutions. II. Diffusion.

We discuss diffusion in micellar surfactant solutions in a form appropriate for analyzing experiments that involve large deviations from equilibrium. A general nonlinear dynamical model for inhomogeneous systems is developed that describes the effects of diffusion and micelle kinetics as a set of coupled partial differential equations for unimer concentration, micelle number concentration, average micelle aggregation number, and, optionally, the variance of the micelle aggregation number. More specialized models are developed to describe slow dynamics in situations in which the system stays in a state of partial local equilibrium or full local equilibrium. As an illustrative example of a nonlinear transport phenomenon, we discuss a simple model of diffusion from an initially homogeneous micellar solution to a rapidly created absorbing interface with fast unimer adsorption.

Nonlinear dynamics in micellar surfactant solutions. I. Kinetics.

This is the first of a pair of articles that present the theory of kinetic and transport phenomena in micelle-forming surfactant solutions in a form that facilitates discussion of large deviations from equilibrium. Our goal is to construct approximate but robust reduced models for both homogeneous and inhomogeneous systems as differential equations for unimer concentration c_{1}, micelle number concentration c_{m}, average micelle aggregation number q and (optionally) aggregation number variance σ_{m}^{2}. This first article discusses kinetics in homogeneous solutions. We focus particularly on developing models that can describe both weakly perturbed states and states in which c_{1} is suppressed significantly below the critical micelle concentration, which leads to rapid shrinkage and dissociation of any remaining micelles. This focus is motivated by the strong local suppression of c_{1} that is predicted to occur near interfaces during some adsorption processes that are considered in the second article. Toward this end, we develop a general nonlinear theory of fast stepwise processes for systems that may be subjected to large changes in q and c_{1}. This is combined with the existing nonlinear theory of slow association and dissociation processes to construct a general model for systems governed by stepwise reaction kinetics. We also consider situations in which the slow process of micelle creation and destruction instead occurs primarily by micelle fission and fusion, and analyze the dependencies of micelle lifetime and the slow relaxation time upon surfactant concentration in systems controlled by either association-dissociation or fission-fusion mechanisms.

A DINOFLAGELLATE TBP-LIKE FACTOR ACTIVATES TRANSCRIPTION FROM A TTTT-BOX IN YEAST.

Dinoflagellates do not have a typical TATA-binding protein (TBP), a subunit of the general transcription factor TFIID complex. Instead, they have a TBP-like factor (TLF) that has been shown to bind TTTT instead of TATA in vitro. The ability of TLF to act as a functional replacement of TBP in vivo has never been assessed, however. Here, we show that a dinoflagellate TLF can drive expression of a reporter gene controlled by a budding yeast promoter whose TATA box was mutated to TTTT. TLF is thus able to bind and activate the yeast RNA polymerase and appear to function normally in the TFIID complex.

Evaluation of Pericardial Tissues from Assorted Species as a Tissue-Engineered Heart Valve Material.

Decellularized pericardial tissue is a strong candidate for a TEHV material as ECM is present to guide cellular infiltration and fixed porcine and bovine pericardial tissue have existing use in bioprosthetic heart valves. In this work, we compare the mechanical and microstructural properties of decellularized-sterilized (DS) porcine, bovine, and bison pericardial tissues with respect to use as a TEHV. H&E staining was used to verify removal of cellular content post-decellularization and to evaluate collagen fiber structure. Additionally, uniaxial and biaxial tension testing were used to compare mechanical performance and, for the latter, acquire constitutive model parameters for subsequent finite element (FE) modeling. H&E staining revealed complete removal of cellular content and good collagen fiber structure. Tensile testing showed comparable mechanical strength between the three DS pericardial tissues and considerably stronger mechanical properties compared to native tissues. Bovine and bison DS pericardial tissues showed the strongest mechanical performance in the FE models with bison demonstrating the overall best mechanical characteristics. The increased thickness of bovine and bison tissues coupled with the strong mechanical behavior and ECM structure indicates that these materials will be resistant to damage until sufficient cellular infiltration has occurred such that damaged tissue can be repaired.

Circulating progenitor cells are associated with plaque progression and long-term outcomes in heart transplant patients.

AIMS: Circulating progenitor cells (CPCs) play a role in vascular repair and plaque stability, while osteocalcin (OC) expressing CPCs have been linked to unstable plaque and adverse cardiovascular outcomes. However, their role in cardiac allograft vasculopathy (CAV) has not been elucidated. This cohort study aimed to investigate the contribution of CPCs on CAV progression and cardiovascular events after heart transplantation. METHODS AND RESULTS: A total of 80 heart transplant patients (mean age 55 ± 14 years, 72% male) undergoing annual intravascular ultrasound (IVUS) had fresh CPCs marked by CD34, CD133, and OC counted in peripheral blood using flow cytometry, on the same day as baseline IVUS. CAV progression was assessed by IVUS as the change (Δ) in plaque volume divided by segment length (PV/SL), adjusted for the time between IVUS measurements [median 3.0, interquartile range (2.8-3.1) years] and was defined as ΔPV/SL that is above the median ΔPV/SL of study population. Major adverse cardiac events (MACEs) were defined as any incident of revascularization, myocardial infarction, heart failure admission, re-transplantation, stroke, and death. Patients with higher CD34+CD133+ CPCs had a decreased risk of CAV progression [odds ratio 0.58, 95% confidence interval (CI) (0.37-0.92), P = 0.01] and MACE [hazard ratio (HR) 0.79, 95% CI (0.66-0.99), P = 0.05] during a median (interquartile range) follow-up of 8.0 years (7.2-8.3). Contrarily, higher OC+ cell counts were associated with an increased risk of MACE [HR 1.26, 95% CI (1.03-1.57), P = 0.02]. CONCLUSIONS: Lower levels of CD34+CD133+ CPCs are associated with plaque progression and adverse long-term outcomes in patients who underwent allograft heart transplantation. In contrast, higher circulating OC+ levels are associated with adverse long-term outcomes. Thus, CPCs might play a role in amelioration of transplant vasculopathy, while OC expression by these cells might play a role in progression.

Carotid Plaques From Symptomatic Patients With Mild Stenosis Is Associated With Intraplaque Hemorrhage.

Carotid plaque vulnerability features beyond the degree of stenosis may play a key role in the pathogenesis and recurrence of ischemic cerebrovascular events. This study sought to compare intraplaque hemorrhage (IPH) as a marker of plaque vulnerability in symptomatic patients with mild (<50%), moderate (50%-69%), and severe (≥70%) carotid artery stenosis. We included patients who experienced ischemic cerebrovascular events with no other identifiable sources and underwent carotid endarterectomy for mild (n=32), moderate (n=47), and severe (n=58) carotid artery stenosis. The degree of stenosis and imaging hallmarks were assessed by computed tomography angiography or magnetic resonance angiography. Plaque specimens were stained with hematoxylin and eosin and Movat pentachrome staining. Carotid plaques of patients with mild stenosis had a higher extent of IPH (%) on tissue analysis compared with patients with moderate (mild, 15.7% [interquartile range, 7.8%-26.7%]; moderate, 3.9% [0.0%-9.2%]; P<0.001) and severe carotid artery stenosis (mild, 15.7% [interquartile range, 7.8%-26.7%]; severe, 2.5% [interquartile range, 0.0%-11.2%]; P<0.001). When considering the degree of carotid artery stenosis as a continuous variable, a lower lumen narrowing was associated with higher extent of IPH (P<0.001; R, -0.329). Our major finding is the association of IPH with mild carotid artery stenosis based on histological analysis. The current study may suggest that IPH potentially plays a role in the mechanism of stroke in patients with nonobstructive carotid stenosis.

Spatial organization of dinoflagellate genomes: Novel insights and remaining critical questions.

As is true for many other aspects, genome architecture, evolution, and function in dinoflagellates are enigmatic and, in the meantime, continuous inspiration for scientific quests. Recent third-generation sequencing and Hi-C linkage analyses brought new insights into the spatial organization of symbiodiniacean genomes, revealing the topologically associated domains, discrete gene clusters and their cis and trans orientations, and relationships with transcription. Where do these new findings bring us in dinoflagellate genomics? Here, we aim to place these new results in the backdrop of the long history of research on this topic and in the context of what critical questions remain to be pursued in the future. The new data suggest, pending verification of other complete chromosome assemblies, a potential evolutionary trend in chromosome number decrease and length increase within the Symbiodiniaceae. While questions remain about the mechanics of the three-dimensional chromosome structure and cell cycle-related DNA replication, the mechanisms of gene transcription and genome size evolution, these latest findings set new starting points for further inquiries.

Assessing nucleic acid binding activity of four dinoflagellate cold shock domain proteins from Symbiodinium kawagutii and Lingulodinium polyedra.

BACKGROUND: Dinoflagellates have a generally large number of genes but only a small percentage of these are annotated as transcription factors. Cold shock domain (CSD) containing proteins (CSPs) account for roughly 60% of these. CSDs are not prevalent in other eukaryotic lineages, perhaps suggesting a lineage-specific expansion of this type of transcription factors in dinoflagellates, but there is little experimental data to support a role for dinoflagellate CSPs as transcription factors. Here we evaluate the hypothesis that dinoflagellate CSPs can act as transcription factors by binding double-stranded DNA in a sequence dependent manner. RESULTS: We find that both electrophoretic mobility shift assay (EMSA) competition experiments and selection and amplification binding (SAAB) assays indicate binding is not sequence specific for four different CSPs from two dinoflagellate species. Competition experiments indicate all four CSPs bind to RNA better than double-stranded DNA. CONCLUSION: Dinoflagellate CSPs do not share the nucleic acid binding properties expected for them to function as bone fide transcription factors. We conclude the transcription factor complement of dinoflagellates is even smaller than previously thought suggesting that dinoflagellates have a reduced dependance on transcriptional control compared to other eukaryotes.

Lipophilicity Determines Routes of Uptake and Clearance, and Toxicity of an Alpha-Particle-Emitting Peptide Receptor Radiotherapy.

Lipophilicity is explored in the biodistribution (BD), pharmacokinetics (PK), radiation dosimetry (RD), and toxicity of an internally administered targeted alpha-particle therapy (TAT) under development for the treatment of metastatic melanoma. The TAT conjugate is comprised of the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate), conjugated to melanocortin receptor 1 specific peptidic ligand (MC1RL) using a linker moiety and chelation of the 225Ac radiometal. A set of conjugates were prepared with a range of lipophilicities (log D 7.4 values) by varying the chemical properties of the linker. Reported are the observations that higher log D 7.4 values are associated with decreased kidney uptake, decreased absorbed radiation dose, and decreased kidney toxicity of the TAT, and the inverse is observed for lower log D 7.4 values. Animals administered TATs with lower lipophilicities exhibited acute nephropathy and death, whereas animals administered the highest activity TATs with higher lipophilicities lived for the duration of the 7 month study and exhibited chronic progressive nephropathy. Changes in TAT lipophilicity were not associated with changes in liver uptake, dose, or toxicity. Significant observations include that lipophilicity correlates with kidney BD, the kidney-to-liver BD ratio, and weight loss and that blood urea nitrogen (BUN) levels correlated with kidney uptake. Furthermore, BUN was identified as having higher sensitivity and specificity of detection of kidney pathology, and the liver enzyme alkaline phosphatase (ALKP) had high sensitivity and specificity for detection of liver damage associated with the TAT. These findings suggest that tuning radiopharmaceutical lipophilicity can effectively modulate the level of kidney uptake to reduce morbidity and improve both safety and efficacy.