Optical diffraction tomography for assessing single cell models in angular light scattering.

Angularly resolved light scattering (ALS) has become a useful tool for assessing the size and refractive index of biological scatterers at cellular and organelle length scales. Sizing organelle populations with ALS relies on Mie scattering theory models, which require significant assumptions about the object, including spherical scatterers and a homogeneous medium. These assumptions may incur greater error at the single cell level, where there are fewer scatterers to be averaged over. We investigate the validity of these assumptions using 3D refractive index (RI) tomograms measured via optical diffraction tomography (ODT). We compute the angular scattering on digitally manipulated tomograms with increasingly strong model assumptions, including RI-matched immersion media, homogeneous cytosol, and spherical organelles. We also compare the tomogram-computed angular scattering to experimental measurements of angular scattering from the same cells to ensure that the ODT-based approach accurately models angular scattering. We show that enforced RI-matching with the immersion medium and a homogeneous cytosol significantly affects the angular scattering intensity shape, suggesting that these assumptions can reduce the accuracy of size distribution estimates.

Three-dimensional angular scattering simulations inform analysis of scattering from single cells.

Significance: Organelle sizes, which are indicative of cellular status, have implications for drug development and immunology research. At the single cell level, such information could be used to study the heterogeneity of cell response to drugs or pathogens. Aim: Angularly resolved elastic light scattering is known to be sensitive to changes in organelle size distribution. We developed a Mie theory-based simulation of angular scattering from single cells to quantify the effects of noise on scattering and size estimates. Approach: We simulated randomly sampled organelle sizes (drawn from a log normal distribution), interference between different organelles' scattering, and detector noise. We quantified each noise source's effect upon the estimated mean and standard deviation of organelle size distributions. Results: The results demonstrate that signal-to-noise ratio in the angular scattering increased with the number of scatterers, cell area, and exposure time and decreased with the size distribution width. The error in estimating the mean of the size distributions remained below 5% for nearly all experimental parameters tested, but the widest size distribution tested (standard deviation of 600 nm) reached 20%. Conclusions: The simulator revealed that sparse sampling of a broad size distribution can dominate the mismatch between actual and predicted size parameters. Alternative estimation strategies could reduce the discrepancy.

Effect of Physical Activity During Chemotherapy on Cognitive Function in Cancer Survivors: A Systematic Review and Meta-Analysis.

Purpose: To determine if cancer survivors who perform physical activity (PA) during chemotherapy have improved levels of cognitive function compared to those who do not. Method: E-databases (Ovid MEDLINE, Embase, CINAHL, PsycINFO, AMED) were searched from inception to February 4, 2020. Quantitative studies that assessed cognitive outcomes for adults with any cancer type who received chemotherapy concurrent with PA were selected. Risk of bias was assessed using Cochrane's RoB 2, ROBINS-I, and Newcastle-Ottawa scales. A meta-analysis was performed using standardized mean difference (SMD). Results: Twenty-two studies (15 randomized controlled trials [RCTs] and 7 non-RCTs) met the inclusion criteria. The meta-analysis demonstrated that combined resistance and aerobic training had a small yet statistically significant effect on social cognition compared to usual care (SMD 0.23 [95% CI: 0.04, 0.42], p = 0.020). Conclusions: Combined resistance and aerobic exercise may benefit social cognition in cancer survivors undergoing chemotherapy. Due to high risk of bias and low quality of evidence of included studies, we recommend further investigation to support these findings and make specific PA recommendations.

Objectif : déterminer si les survivants du cancer qui font de l'activité physique (AP) pendant la chimiothérapie ont une meilleure fonction cognitive que ceux qui n'en font pas. Méthodologie : les chercheurs ont fouillé des bases de données électroniques (Ovid MEDLINE, Embase, CINAHL, PsycINFO, AMED) à compter de leur création jusqu'au 4 avril 2020. Ils ont sélectionné les études quantitatives qui évaluaient les issues cognitives des adultes atteints de quelque type de cancer que ce soit et qui avaient été sous chimiothérapie tout en faisant de l'AP. Ils ont évalué le risque de biais au moyen des échelles RoB 2 et ROBINS-I de Cochrane et de l'échelle de Newcastle-Ottawa et ont effectué une méta-analyse au moyen de la différence moyenne standardisée (DMS). Résultats : au total, 22 études (15 essais cliniques randomisés [ÉCR] et sept essais cliniques non randomisés [non-ÉCR]) respectaient les critères d'inclusion. La méta-analyse a démontré que la combinaison d'exercices de résistance et d'exercices aérobiques avait un effet statistique petit, mais significatif, sur la cognition sociale par rapport aux soins habituels (DMS = 0,23 [IC a 95 % : 0,04, 0,42], p = 0,020). Conclusions : la combinaison d'exercices de résistance et d'exercices aérobiques peut être bénéfique à la cognition sociale des survivants du cancer sous chimiothérapie. Étant donné le risque élevé de biais et la faible qualité des données probantes des études retenues, les chercheurs recommandent de poursuivre les recherches pour appuyer ces résultats et faire des recommandations particulières en matière d'AP.

Matching an immersion medium's refractive index to a cell's cytosol isolates organelle scattering.

Angularly-resolved light scattering has been proven to be an early detector of subtle changes in organelle size due to its sensitivity to scatterer size and refractive index contrast. However, for cells immersed in media with a refractive index close to 1.33, the cell itself acts as a larger scatterer and contributes its own angular signature. This whole-cell scattering, highly dependent on the cell's shape and size, is challenging to distinguish from the desired organelle scattering signal. This degrades the accuracy with which organelle size information can be extracted from the angular scattering. To mitigate this effect, we manipulate the refractive index of the immersion medium by mixing it with a water-soluble, biocompatible, high-refractive-index liquid. This approach physically reduces the amount of whole-cell scattering by minimizing the refractive index contrast between the cytosol and the modified medium. We demonstrate this technique on live cells adherent on a coverslip, using Fourier transform light scattering to compute the angular scattering from complex field images. We show that scattering from the cell: media refractive index contrast contributes significant scattering at angles up to twenty degrees and that refractive index-matching reduces such low-angle scatter by factors of up to 4.5. This result indicates the potential of refractive index-matching for improving the estimates of organelle size distributions in single cells.

Direct coculture of human pluripotent stem cell-derived cardiac progenitor cells with epicardial cells induces cardiomyocyte proliferation and reduces sarcomere organization.

Epicardial cells (EpiCs) are necessary for myocardium formation, yet little is known about crosstalk between EpiCs and cardiomyocytes (CMs) during development and the potential impact of EpiCs on CM maturation. To investigate the effects of EpiCs on CM commitment and maturation, we differentiated human pluripotent stem cells (hPSCs) to cardiac progenitor cells (CPCs) and EpiCs, and cocultured EpiCs and CPCs for two weeks. When EpiCs were allowed to form epicardial-derived cells, we observed increased expression of cTnI in developing CMs. In the presence of the TGFß inhibitor A83-01, EpiCs remained in the epicardial state and induced CM proliferation, increased MLC2v expression, and led to less organized sarcomeres. These effects were not observed if CPCs were treated with EpiC-conditioned medium or if CPCs were indirectly cocultured with EpiCs. Finally, single cell RNA sequencing identified that EpiC-CPC coculture had bi-directional effects on transcriptional programs in EpiCs and CMs, and biased EpiC lineages from a SFRP2-enriched population to a DLK1- or C3-enriched population. This work suggests important crosstalk between EpiCs and CMs during differentiation which can be used to influence cell fate and improve the ability to generate cardiac cells and tissues for in vitro models and development of cardiac cellular therapies.

Phase-sensitive, angle-resolved light-scattering microscopy of single cells.

We report what is to our knowledge the first use of Fourier phase microscopy (FPM) to estimate diameters of individual single-micrometer beads and to classify cells based upon changes in scatterer size distribution. FPM, a quantitative phase imaging (QPI) method, combines the planar illumination typically used in off-axis QPI (ideal for Mie theory analysis) with the common-path geometry typically used in on-axis QPI (ideal for optimizing angular scattering range). Low-spatial-frequency imaging artifacts inherent to FPM have negligible impact upon these angular-domain applications. The system is simple to align and stable, and requires no external reference beam. Angular scattering patterns obtained from single 1 µm polystyrene beads in glycerol (Δn=0.11) display unprecedented fidelity to Mie theory, produce diameter estimates consistent with the manufacturer's specifications, and offer precision on the scale of tens of nanometers. Measurements of macrophages at different stages of antibody-dependent cellular phagocytosis demonstrate the ability to detect changes in a cell's scattering caused by the presence of phagocytosed material within.

Small-Molecule Morphogenesis Modulators Enhance the Ability of 14-Helical ß-Peptides To Prevent Candida albicans Biofilm Formation.

Candida albicans is an opportunistic fungal pathogen responsible for mucosal candidiasis and systemic candidemia in humans. Often, these infections are associated with the formation of drug-resistant biofilms on the surfaces of tissues or medical devices. Increased incidence of C. albicans resistance to current antifungals has heightened the need for new strategies to prevent or eliminate biofilm-related fungal infections. In prior studies, we designed 14-helical ß-peptides to mimic the structural properties of natural antimicrobial α-peptides (AMPs) in an effort to develop active and selective antifungal compounds. These amphiphilic, cationic, helical ß-peptides exhibited antifungal activity against planktonic C. albicans cells and inhibited biofilm formation in vitro and in vivo Recent studies have suggested the use of antivirulence agents in combination with antifungals. In this study, we investigated the use of compounds that target C. albicans polymorphism, such as 1-dodecanol, isoamyl alcohol, and farnesol, to attempt to improve ß-peptide efficacy for preventing C. albicans biofilms. Isoamyl alcohol, which prevents hyphal formation, reduced the minimum biofilm prevention concentrations (MBPCs) of ß-peptides by up to 128-fold. Combinations of isoamyl alcohol and antifungal ß-peptides resulted in less than 10% hemolysis at the antifungal MBPCs. Overall, our results suggest potential benefits of combination therapies comprised of morphogenesis modulators and antifungal AMP peptidomimetics for preventing C. albicans biofilm formation.

Coculture of Endothelial Cells with Human Pluripotent Stem Cell-Derived Cardiac Progenitors Reveals a Differentiation Stage-Specific Enhancement of Cardiomyocyte Maturation.

Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are immature in their structure and function, limiting their potential in disease modeling, drug screening, and cardiac cellular therapies. Prior studies have demonstrated that coculture of hPSC-derived CMs with other cardiac cell types, including endothelial cells (ECs), can accelerate CM maturation. To address whether the CM differentiation stage at which ECs are introduced affects CM maturation, the authors coculture hPSC-derived ECs with hPSC-derived cardiac progenitor cells (CPCs) and CMs and analyze the molecular and functional attributes of maturation. ECs have a more significant effect on acceleration of maturation when cocultured with CPCs than with CMs. EC coculture with CPCs increases CM size, expression of sarcomere, and ion channel genes and proteins, the presence of intracellular membranous extensions, and chronotropic response compared to monoculture. Maturation is accelerated with an increasing EC:CPC ratio. This study demonstrates that EC incorporation at the CPC stage of CM differentiation expedites CM maturation, leading to cells that may be better suited for in vitro and in vivo applications of hPSC-derived CMs.

Engineering Scalable Manufacturing of High-Quality Stem Cell-Derived Cardiomyocytes for Cardiac Tissue Repair.

Recent advances in the differentiation and production of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) have stimulated development of strategies to use these cells in human cardiac regenerative therapies. A prerequisite for clinical trials and translational implementation of hPSC-derived CMs is the ability to manufacture safe and potent cells on the scale needed to replace cells lost during heart disease. Current differentiation protocols generate fetal-like CMs that exhibit proarrhythmogenic potential. Sufficient maturation of these hPSC-derived CMs has yet to be achieved to allow these cells to be used as a regenerative medicine therapy. Insights into the native cardiac environment during heart development may enable engineering of strategies that guide hPSC-derived CMs to mature. Specifically, considerations must be made in regard to developing methods to incorporate the native intercellular interactions and biomechanical cues into hPSC-derived CM production that are conducive to scale-up.

Experimental measurements of the magnitude and phase response of high-frequency modulated light underwater.

The propagation behavior of high-frequency intensity-modulated signals through turbid water is of significant interest for underwater laser ranging, imaging, and communications. Prior experimental measurements have focused only on the magnitude response of the underwater optical channel to forward-scattered and unscattered modulated light. In this study we include, for the first time to our knowledge, both the magnitude and phase of the underwater optical channel to forward-scattered light. The magnitude and phase response is measured out to 1 GHz, using three different artificial scattering agents in scattering environments in excess of 25 attenuation lengths. The phase response provides additional insight into the behavior of forward-scattered light carrying high-frequency intensity modulation.

Metabolomics Identifies Metabolic Markers of Maturation in Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiovascular disease is a leading cause of death worldwide. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold immense clinical potential and recent studies have enabled generation of virtually pure hPSC-CMs with high efficiency in chemically defined and xeno-free conditions. Despite these advances, hPSC-CMs exhibit an immature phenotype and are arrhythmogenic in vivo, necessitating development of strategies to mature these cells. hPSC-CMs undergo significant metabolic alterations during differentiation and maturation. A detailed analysis of the metabolic changes accompanying maturation of hPSC-CMs may prove useful in identifying new strategies to expedite hPSC-CM maturation and also may provide biomarkers for testing or validating hPSC-CM maturation. In this study we identified global metabolic changes which take place during long-term culture and maturation of hPSC-CMs derived from three different hPSC lines. We have identified several metabolic pathways, including phospholipid metabolism and pantothenate and Coenzyme A metabolism, which showed significant enrichment upon maturation in addition to fatty acid oxidation and metabolism. We also identified increases in glycerophosphocholine and the glycerophosphocholine:phosphocholine ratio as potential metabolic biomarkers of maturation. These biomarkers were also affected in a similar manner during murine heart development in vivo. These results support that hPSC-CM maturation is associated with extensive metabolic changes in metabolic network utilization and understanding the roles of these metabolic changes has the potential to develop novel approaches to monitor and expedite hPSC-CM maturation.

Propagation of modulated optical beams carrying orbital angular momentum in turbid water.

The attenuation and temporal dispersion of beams with and without orbital angular momentum (OAM) underwater are investigated in a controlled laboratory water tank environment. Both spherical polystyrene beads and a commercial antacid are used to determine the effect of scattering particle size and shape on the results. Varying concentrations of the scattering agents were used to study the propagation of light in both minimally scattered and multiply scattered regimes (over 20 attenuation lengths). To study temporal dispersion, a custom diode seeded fiber amplified laser source is used to modulate beams up to 1 GHz, and diffractive spiral phase plates are used to compare performance over different spatial modes. We observe an increase in received signal with increasing OAM order (|m|=0, 8, and 16) under multiple scattering conditions. Initial experimental results suggest that this variation is dependent on particle shape and size. We do not observe any dependency of OAM order on temporal dispersion.

Chemically-defined albumin-free differentiation of human pluripotent stem cells to endothelial progenitor cells.

Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors are important for vascular research and therapeutic revascularization. Here, we report a completely defined endothelial progenitor differentiation platform that uses a minimalistic medium consisting of Dulbecco's modified eagle medium and ascorbic acid, lacking of albumin and growth factors. Following hPSC treatment with a GSK-3ß inhibitor and culture in this medium, this protocol generates more than 30% multipotent CD34+ CD31+ endothelial progenitors that can be purified to >95% CD34+ cells via magnetic activated cell sorting (MACS). These CD34+ progenitors are capable of differentiating into endothelial cells in serum-free inductive media. These hPSC-derived endothelial cells express key endothelial markers including CD31, VE-cadherin, and von Willebrand factor (vWF), exhibit endothelial-specific phenotypes and functions including tube formation and acetylated low-density lipoprotein (Ac-LDL) uptake. This fully defined platform should facilitate production of proliferative, xeno-free endothelial progenitor cells for both research and clinical applications.

The ongoing cognitive processing of exclusionary social events: evidence from event-related potentials.

Exclusionary social events are known to cause alterations in neural activity and attention-related processes. However, the precise nature of these neural adjustments remains unknown as previous research has been limited to examining social interactions and exclusionary events as unitary phenomena. To address this limitation, we assessed neural activity during both inclusionary and exclusionary social interactions by examining event-related brain potentials at multiple points within each social event. Our results show an initial enhancement of anterior cingulate cortex -related activation, indexed by the anterior N2, in response to specific exclusionary events followed by an enhanced attentional orienting response, indexed by the P3a, to later segments of each exclusionary event. Decreases in this P3a activation from social inclusion to social exclusion were associated with self-reported increases in anxiety, negative affect, and feelings of depression from inclusion to exclusion. Together, these findings provide novel insights into the dynamic and ongoing neural processes associated with attentional allocation toward social exclusion and the nature of the relationships between neural and behavioral reactions to exclusionary social interactions.

Exploiting post-transcriptional regulation to probe RNA structures in vivo via fluorescence.

While RNA structures have been extensively characterized in vitro, very few techniques exist to probe RNA structures inside cells. Here, we have exploited mechanisms of post-transcriptional regulation to synthesize fluorescence-based probes that assay RNA structures in vivo. Our probing system involves the co-expression of two constructs: (i) a target RNA and (ii) a reporter containing a probe complementary to a region in the target RNA attached to an RBS-sequestering hairpin and fused to a sequence encoding the green fluorescent protein (GFP). When a region of the target RNA is accessible, the area can interact with its complementary probe, resulting in fluorescence. By using this system, we observed varied patterns of structural accessibility along the length of the Tetrahymena group I intron. We performed in vivo DMS footprinting which, along with previous footprinting studies, helped to explain our probing results. Additionally, this novel approach represents a valuable tool to differentiate between RNA variants and to detect structural changes caused by subtle mutations. Our results capture some differences from traditional footprinting assays that could suggest that probing in vivo via oligonucleotide hybridization facilitates the detection of folding intermediates. Importantly, our data indicate that intracellular oligonucleotide probing can be a powerful complement to existing RNA structural probing methods.

Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of WNT signaling.

Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. Here, we report a method to efficiently produce endothelial cells from hPSCs via GSK3 inhibition and culture in defined media to direct hPSC differentiation to CD34(+)CD31(+) endothelial progenitors. Exogenous vascular endothelial growth factor (VEGF) treatment was dispensable, and endothelial progenitor differentiation was ß-catenin dependent. Furthermore, by clonal analysis, we showed that CD34(+)CD31(+)CD117(+)TIE-2(+) endothelial progenitors were multipotent, capable of differentiating into calponin-expressing smooth muscle cells and CD31(+)CD144(+)vWF(+)I-CAM1(+) endothelial cells. These endothelial cells were capable of 20 population doublings, formed tube-like structures, imported acetylated low-density lipoprotein, and maintained a dynamic barrier function. This study provides a rapid and efficient method for production of hPSC-derived endothelial progenitors and endothelial cells and identifies WNT/ß-catenin signaling as a primary regulator for generating vascular cells from hPSCs.