Development of a flow cytometry–based pulse-width assay for detection of aggregates in cellular therapeutics to be infused by catheter

Background aimsDelivery of cell-based therapies through the carotid artery with the use of an intra-arterial catheter could introduce aggregates and cause focal ischemia in the brain. We developed a pulse-width flow cytometry method for aggregate detection and quantification. The assay was designed to be used as a cell product release assay in a clinical trial seeking to treat ischemic stroke with sorted cells brightly expressing aldehyde dehydrogenase (ALDH^br cells) delivered through intra-arterial catheters.MethodsThe forward light scatter pulse-width axis of a flow cytometer was calibrated for particle diameter measurements through the use of traceable standard microspheres and linear regression. As a positive control, Concanavalin A–aggregated cells were counted manually and sorted onto slides to compare with pulse width–determined values. Known numbers of aggregates were spiked into purified singlet cells for quantification. A clinical standard for aggregate count and diameter was determined. The assay was used to qualify catheters with the use of ALDH^br cells.ResultsThe pulse-width axis was highly linear for microsphere diameter (r² > 0.99), which allowed for size calibration. Microscopically determined counts and diameters corresponded to pulse width-determined values. Known aggregate counts were linear with pulse width–determined aggregate counts (r² = 0.98). The limit of detection was determined to be 0.004%. Flow of ALDH^br cells through catheters did not generate aggregates. The final method to be used as a release assay for the stroke clinical trial was tested successfully on samples from volunteer donors.ConclusionsThe pulse-width aggregate detection assay provides a reliable, reproducible, accurate and rapid means of detection, classification and quantification of aggregates in cell therapy products.     

Comparison of natural and pharmacological hypothermia in animals: Determination of activation energy of metabolism

The constancy of the activation energy of metabolism (E) for all living organisms is one of the most impressive, though controversial, statements of the modern metabolic theory of evolution. According to WBE-theory suggested by West, Brown, and Enquist, E should be in the range from −0.6 to −0.7 eV. However, there are many examples of significant deviations of E from the predictions of the theory. Now we have conducted a study of this value using rats in different types of pharmacological hypothermia: 1. Short-term (for several hours) hypothermia induced by anesthetic xylazine; 2. Daily torpor-like state induced by the pharmacological composition developed in our previous study. It has been found that in pharmacological daily hypothermia E = −0.56 ± 0.03 eV, which was close to that in daily heterotherms found in literature, E = −0.57 ± 0.04 eV. In short-term hypothermia E was substantially lower, E = −0.17 ± 0.071 eV. Our analysis revealed that in short-term hypothermia, changes in body temperature may lag behind changes in metabolic rate for a period Δt, affecting E. We propose an approach for estimating Δt and obtaining an adjusted E = −0.68 ± 0.17 eV, which corresponds to theoretical predictions. We assume that a similar consideration of Δt should be done when calculating E of daily heterotherms. We assume that in ectotherms, when the ambient temperature changes rapidly, changes in metabolic rate may lag behind changes in body temperature for a period (−) Δt, that should also be considered in E calculations. The proposed approach may contribute to the further development of the metabolic theory of evolution and may be useful in comparing artificial and natural hypothermia, as well as in studying the energy transformations in ecosystems.     

Trait means or variance—What determines plant species' local and regional occurrence in fragmented dry grasslands?

One of the few laws in ecology is that communities consist of few common and many rare taxa. Functional traits may help to identify the underlying mechanisms of this community pattern, since they correlate with different niche dimensions. However, comprehensive studies are missing that investigate the effects of species mean traits (niche position) and intraspecific trait variability (ITV, niche width) on species abundance. In this study, we investigated fragmented dry grasslands to reveal trait‐occurrence relationships in plants at local and regional scales. We predicted that (a) at the local scale, species occurrence is highest for species with intermediate traits, (b) at the regional scale, habitat specialists have a lower species occurrence than generalists, and thus, traits associated with stress‐tolerance have a negative effect on species occurrence, and (c) ITV increases species occurrence irrespective of the scale. We measured three plant functional traits (SLA = specific leaf area, LDMC = leaf dry matter content, plant height) at 21 local dry grassland communities (10 m × 10 m) and analyzed the effect of these traits and their variation on species occurrence. At the local scale, mean LDMC had a positive effect on species occurrence, indicating that stress‐tolerant species are the most abundant rather than species with intermediate traits (hypothesis 1). We found limited support for lower specialist occurrence at the regional scale (hypothesis 2). Further, ITV of LDMC and plant height had a positive effect on local occurrence supporting hypothesis 3. In contrast, at the regional scale, plants with a higher ITV of plant height were less frequent. We found no evidence that the consideration of phylogenetic relationships in our analyses influenced our findings. In conclusion, both species mean traits (in particular LDMC) and ITV were differently related to species occurrence with respect to spatial scale. Therefore, our study underlines the strong scale‐dependency of trait‐abundance relationships.     

Dendroclimatic signals in the pine and spruce chronologies in the Solovetsky Archipelago

Searching for a robust tree-ring parameter useful for paleoclimatic purposes is one of the most demanding topics in the modern paleoscience. Since Blue Intensity has already expressed itself in different geographical locations all over the world as a possible replacement for maximum density, close attention is paid to investigate features of the inferred signal. The Solovki Islands is a unique location in Northern Russia where two important factors that make this territory attractive for developing a long tree-ring chronology have been met: modern long-living trees and building activities using old trees that were started by monks in the middle of the 16th century. The main goal of the research is to develop pine and spruce chronologies based on tree-ring width (TRW) and delta Blue Intensity (dBI) and to assess the ability of these parameters to be used as climate predictors. As a result, 14 conifer chronologies from 7 sites (4 for pine and 3 for spruce) were developed. The composite pine and spruce chronologies span a period of 474 and 378 years each. Cross-correlation of dBI-based chronologies of both conifers is high (r = up to 0.71 while for TRW-based chronologies it is lower on average (−0.18 to 0.63). Intra-species correlation of TRW chronologies in some cases achieved even negative values (r = −0.18. Discrepancies found between TRW chronologies of pine and spruce could be explained by differences in climatic signals. Response function analysis with monthly temperatures revealed that growth of pine depends on the previous August, while spruce has a temporally stable and strong relation to June temperatures. Compared to TRW, dBI-based chronologies have a high correlation with summer temperatures (r = 0.64 and 0.66 for spruce and pine, respectively). Presented research points out the importance of the response function analysis suggesting that depending on goals of the study several tree-ring parameters could be used, e.g., tree-ring width of spruce responses to June temperatures, while dBI to the whole summer.     

How do babies know their friends and foes?

The study of infant social cognition is the study of how human infants acquire information about people. By examining infants’ sensory abilities and the stimulus characteristics of people, research can determine what information is available to infants from their social world. We can then consider what social environments are appropriate for infants of different ages. This paper examines the sociocognitive competencies of human infants during the first 6 months of their lives and asks how these competencies are functional in the daily social ecology of the human infant. Select examples of research with other species are used to illustrate how the adaptive significance of sociocognitive abilities could be more fruitfully explored in studies of human infancy. Lonnie R. Sherrod is Vice President for Program at the William T. Grant Foundation. Formerly, he was Assistant Dean at the Graduate Faculty of the New School for Social Research and before that, Staff Associate at the Social Science Research Council. He received a Ph.D. in Developmental Psychology from Yale University in 1978, an M.A. in Biology from the University of Rochester in 1974; and a B.A. in Zoology and Psychology from Duke University in 1972. He has taught at New York University and the New School and has published numerous articles and edited volumes on infant social cognition, on adolescence, and on child development from a life-span and biosocial perspective. Examples includeInfant Social Cognition (1981), edited with Michael Lamb;The Life Course and Human Development: Multidisciplinary Perspectives (1986), edited with Aage B. Sorensen and Franz E. Weinert; and “Changes in Children’s Social Lives and the Development of Social Understanding” authored with Judith Dunn (1988), in E.M. Hetherington, M. Perlmutter, and R. Lerner (eds).,Child Development in Life-Span Perspective.     

Parental preferences for the facial traits of their offspring's partners can enhance parental inclusive fitness

Physical appearance provides a wealth of information concerning an individual's biological fitness and reproductive quality, but we do not know whether parents make use of this information when evaluating potential partners for their offspring. This is critical to our understanding of human mate choice, because parents frequently influence their offspring's mating decisions, either directly, for instance through arranged marriages, or indirectly, through manipulating their offspring's partner choice. Here, we used facial images that varied in attractiveness, masculinity, health, and symmetry to assess both reproductively-aged daughters' and their parents' preferences in potential mates for the daughters. In line with our predictions, both daughters and their parents had clear preferences for markers of genetic quality, although the daughters showed significantly stronger preferences for these markers than their parents. Contrary to previous research, parents and daughters did not have stronger preferences for markers of genetic quality if they perceived the daughter to be more attractive. Parents' preferences for the facial markers of genetic quality in their offspring's partner may help maximize inclusive fitness.     

Amperometric measurements of catecholamine release from single vesicles in MN9D cells

MN9D cells have been used as a successful model to investigate dopamine pharmacology and to test the specific effects of drugs for the treatment of Parkinson’s disease. However, quantitative measurements of quantal release from these cells have not been carried out. In this work, we used amperometry to investigate catecholamine release from MN9D cells. Amperometric events were observed in both undifferentiated and differentiated (butyric acid‐treated) cells. An increase in quantal size and half‐width was observed for differentiated cells versus undifferentiated cells; however, the number of events per cell and the amplitude remained constant. In transmission electron microscopy images, no obvious cluster of small synaptic vesicles was observed, and large dense‐core vesicles were present in the cell body of undifferentiated cells; however, after differentiation, vesicles were concentrated in the cell processes. In differentiated cells, l ‐DOPA caused an increase in quantal size and half‐width, which could be blocked by the vesicular monoamine transporter inhibitor, reserpine.     

The experimental evolution of specialists,generalists, and the maintenance of diversity

Environmental heterogeneity may be a general explanation for both the quantity of genetic variation in populations and the ecological niche width of individuals. To evaluate this hypothesis, I review the literature on selection experiments in heterogeneous environments. The niche width usually – but not invariably – evolves to match the amount of environmental variation, specialists evolving in homogeneous environments and generalists evolving in heterogeneous environments. The genetics of niche width are more complex than has previously been recognized, particularly with respect to the magnitude of costs of adaptation and the putative constraints on the evolution of generalists. Genetic variation in fitness is more readily maintained in heterogeneous environments than in homogeneous environments and this diversity is often stably maintained through negative frequency‐dependent selection. Moreover environmental heterogeneity appears to be a plausible mechanism for at least two well‐known patterns of species diversity at the landscape scale. I conclude that environmental heterogeneity is a plausible and possibly very general explanation for diversity across the range of scales from individuals to landscapes.