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At least 307 records · Page 17

The Effects of Spaceflight on Cellular Aging in Saccharomyces cerevisiae

The conditions encountered during spaceflight place unique stresses on physiological processes that oftentimes lead to deleterious effects. Identifying these effects and better understanding their molecular mechanisms will be essential in enabling long-duration space travel by humans. Studies in Saccharomyces cerevisiae suggest an aging model that involves the accumulation of toxic components, such as excess extrachromosomal rDNA and damaged mitochondria. This build-up then limits the replicative lifespan (the number of times a mother cell can form a new daughter cell). Remarkably, each new daughter cell emerges completely renewed from the senescing mother cell through an asymmetric distribution of aging determinants via mechanisms that are intricately linked to the budding process. When exposed to simulated microgravity, S. cerevisiae undergoes an altered budding process characterized by a breakdown in bud scar polarity. Because the budding process is critical to replicative aging, we hypothesize that the replicative lifespan may be affected by microgravity as well. To measure relative replicative aging rates, we will construct a strain of yeast in which daughter cells are inviable. In this strain, the Cre recombinase will be expressed under the control of the daughter cell specific promoter, pSCW11, and LoxP sites will be inserted at both flanks of two essential genes involved in the cell cycle, UBC9 and CDC20, using a CRISPRCas9 system. Thus, UBC9 and CDC20 will be excised from daughter cells, leading to cell-cycle arrest and eventual death. To mimic the low shear conditions encountered in microgravity, this strain will be grown in rotating wall vessels. The number of viable mother cells will be monitored over time, and this rate will be compared to cells growing in standard conditions. Because asymmetric division also occurs in mammalian cells (e.g. in neural stem cells), this study will provide insight into how cellular aging rates may change in mammals and will help empower humans to thrive in space for extended and even indefinite periods of time.

aging↗

Barcoded reciprocal hemizygosity analysis via sequencing illuminates the complex genetic basis of yeast thermotolerance

Decades of successes in statistical genetics have revealed the molecular underpinnings of traits as they vary across individuals of a given species. But standard methods in the field cannot be applied to divergences between reproductively isolated taxa. Genome-wide reciprocal hemizygosity mapping (RH-seq), a mutagenesis screen in an interspecies hybrid background, holds promise as a method to accelerate the progress of interspecies genetics research. Here, we describe an improvement to RH-seq in which mutants harbor barcodes for cheap and straightforward sequencing after selection in a condition of interest. As a proof of concept for the new tool, we carried out genetic dissection of the difference in thermotolerance between two reproductively isolated budding yeast species. Experimental screening identified dozens of candidate loci at which variation between the species contributed to the thermotolerance trait. Hits were enriched for mitosis genes and other housekeeping factors, and among them were multiple loci with robust sequence signatures of positive selection. Together, these results shed new light on the mechanisms by which evolution solved the problems of cell survival and division at high temperature in the yeast clade, and they illustrate the power of the barcoded RH-seq approach.

59 BASIC BIOLOGICAL SCIENCES↗

Syncytia in Fungi

Filamentous fungi typically grow as interconnected multinucleate syncytia that can be microscopic to many hectares in size. Mechanistic details and rules that govern the formation and function of these multinucleate syncytia are largely unexplored, including details on syncytial morphology and the regulatory controls of cellular and molecular processes. Recent discoveries have revealed various adaptations that enable fungal syncytia to accomplish coordinated behaviors, including cell growth, nuclear division, secretion, communication, and adaptation of the hyphal network for mixing nuclear and cytoplasmic organelles. In this review, we highlight recent studies using advanced technologies to define rules that govern organizing principles of hyphal and colony differentiation, including various aspects of nuclear and mitochondrial cooperation versus competition. We place these findings into context with previous foundational literature and present still unanswered questions on mechanistic aspects, function, and morphological diversity of fungal syncytia across the fungal kingdom.

59 BASIC BIOLOGICAL SCIENCES↗

A study of phycophysiology in controlled environments

The primary objective of this research is to obtain fundamental data concerning the growth and metabolism of the unicellular green algae. These organisms are most likely to provide biological oxygen and a food source for space crews. Biochemical conversions, chemical composition, and cell growth and division are discussed. Chlorella sorokiniana is emphasized.

Krauss, R. W.↗

A synchronous binary array divider

An asynchronous binary divider formed of an array of identical logic cells is described. Each cell includes a single bit binary subtractor and a selection gate. The array is connected to divisor, dividend, quotient and remainder registers. Divisor and dividend numbers are read into the divisor and dividend registers, respectively. The array of identical logic cells performs the division in parallel asynchronously and places the results of the division in the quotient and remainder registers for subsequent readout.

Wang, G. Y.↗

Genotoxic effects of high-energy iron particles in human lymphoblasts differing in radiation sensitivity

The effects of (56)Fe particles and (137)Cs gamma radiation were compared in TK6 and WTK1 human lymphoblasts, two related cell lines which differ in TP53 status and in the ability to rejoin DNA double-strand breaks. Both cell lines were more sensitive to the cytotoxic and clastogenic effects of (56)Fe particles than to those of gamma rays. However, the mutagenicity of (56)Fe particles and gamma rays at the TK locus was the same per unit dose and was higher for gamma rays than for (56)Fe particles at isotoxic doses. The respective RBEs for TK6 and WTK1 cells were 1.5 and 1.9 for cytotoxicity and 2.5 and 1.9 for clastogenicity, but only 1 for mutagenicity. The results indicate that complex lesions induced by (56)Fe particles are repaired less efficiently than gamma-ray-induced lesions, leading to fewer colony-forming cells, a slightly higher proportion of aberrant cells at the first division, and a lower frequency of viable mutants at isotoxic doses. WTK1 cells (mutant TP53) were more resistant to the cytotoxic effects of both gamma rays and (56)Fe particles, but showed greater cytogenetic and mutagenic damage than TK6 cells (TP53(+)). A deficiency in the number of damaged TK6 cells (a) reaching the first mitosis after exposure and (b) forming viable mutants can explain these results.

Non-NASA Center↗

The Biological Effectiveness of Silicon Ions is Significantly Higher than Iron Ions for the Induction of Chromosome Damage in Human Lymphocytes

Chromosome aberrations were measured in human peripheral blood lymphocytes after in vitro exposure to Si-28-ions with energies ranging from 90 to 600 MeV/u, or Fe-56-ions with energies ranging from 200 to 5,000 MeV/u. The LET of the various Fe beams in this study ranged from 145 to 440 keV/micron and the LET Si ions ranged from 48 to 158 keV/micron. Doses delivered were in the 10 to 200 cGy range. Dose response curves for chromosome exchanges in cells at first division after exposure, measured using fluorescence in situ hybridization (FISH) with whole chromosome probes, were fitted with linear or linear-quadratic functions. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose response curve for chromosome damage with respect to gamma-rays. The estimates of RBE(sub max) values for total chromosome exchanges ranged from 4.4+/-0.4 to 31.5+/-2.6 for Fe ions, and 11.8+/-1.0 to 42.2+/-3.3 for Si ions. The highest RBE(sub max) value for Fe ions was obtained with the 600 Mev/u beam and 170 MeV/u beam produced the highest RBE(sub max) value for Si ions. For both ions the RBE(sub max) values increased with LET, reaching a maximum at about 180 keV/micron for Fe and about 100 keV/micron for Si, and decreased with further increase in LET.

George, Kerry↗

The Biological Effectiveness of Different Radiation Qualities for the Induction of Chromosome Damage in Human Lymphocytes

Chromosome aberrations were measured in human peripheral blood lymphocytes after in vitro exposure to 28Si- ions with energies ranging from 90 to 600 MeV/u, or to 56Fe-ions with energies ranging from 200 to 5,000 MeV/u. The LET of the various Fe beams in this study ranged from 145 to 440 keV/micron and the LET of the Si ions ranged from 48 to 158 keV/ m. Doses delivered were in the 10- to 200-cGy range. Dose-response curves for chromosome exchanges in cells at first division after exposure, measured using fluorescence in situ hybridization (FISH) with whole-chromosome probes, were fitted with linear or linear-quadratic functions. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose-response curve for chromosome damage with respect to -rays. The estimates of RBE(sub max) values for total chromosome exchanges ranged from 4.4+/-0.4 to 31.5+/-2.6 for Fe ions, and 11.8+/-1.0 to 42.2+/-3.3 for Si ions. The highest RBE(sub max) value for Fe ions was obtained with the 600-Mev/u beam, and the highest RBE(sub max) value for Si ions was obtained with the 170 MeV/u beam. For both ions the RBEmax values increased with LET, reaching a maximum at about 180 keV/micron for Fe and about 100 keV/ m for Si, and decreasing with further increase in LET. Additional studies for low doses 28Si-ions down to 0.02 Gy will be discussed.

Hada, M.↗

The Biological Effectiveness of Different Radiation Qualities for the Induction of Chromosome Damage in Human Lymphocytes

Chromosome aberrations were measured in human peripheral blood lymphocytes after in vitro exposure to Si-28-ions with energies ranging from 90 to 600 MeV/u, Ti-48-ions with energies ranging from 240 to 1000 MeV/u, or to Fe-56-ions with energies ranging from 200 to 5,000 MeV/u. The LET of the various Si beams in this study ranged from 48 to 158 keV/ m, the LET of the Ti ions ranged from 107 to 240 keV/micron, and the LET of the Fe-ions ranged from 145 to 440 keV/ m. Doses delivered were in the 10- to 200-cGy range. Dose-response curves for chromosome exchanges in cells at first division after exposure, measured using fluorescence in situ hybridization (FISH) with whole-chromosome probes, were fitted with linear or linear-quadratic functions. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose-response curve for chromosome damage with respect to gamma-rays. The estimates of RBEmax values for total chromosome exchanges ranged from 4.4+/-0.4 to 31.5+/-2.6 for Fe ions, 21.4+/-1.7 to 28.3+/-2.4 for Ti ions, and 11.8+/-1.0 to 42.2+/-3.3 for Si ions. The highest RBEmax value for Fe ions was obtained with the 600 MeV/u beam, the highest RBEmax value for Ti ions was obtained 1000 MeV/u beam, and the highest RBEmax value for Si ions was obtained with the 170 MeV/u beam. For Si and Fe ions the RBEmax values increased with LET, reaching a maximum at about 180 keV/micron for Fe and about 100 keV/micron for Si, and decreasing with further increase in LET. Additional studies for low doses Si-28-ions down to 0.02 Gy will be discussed.

Hada, M.↗

The Biological Effectiveness of Four Energies of Neon Ions for the Induction of Chromosome Damage in Human Lymphocytes

Chromosomal aberrations were measured in human peripheral blood lymphocytes after in vitro exposure to neon ions at energies of 64, 89, 142, or 267. The corresponding LET values for these energies of neon ranged from 38-103 keV/micrometers and doses delivered were in the 10 to 80 cGy range. Chromosome exchanges were assessed in metaphase and G2 phase cells at first division after exposure using fluorescence in situ hybridization (FISH) with whole chromosome probes and dose response curves were generated for different types of chromosomal exchanges. The yields of total chromosome exchanges were similar for the 64, 89, and 142 MeV exposures, whereas the 267 MeV/u neon with LET of 38 keV/micrometers produced about half as many exchanges per unit dose. The induction of complex type chromosome exchanges (exchanges involving three or more breaks and two or more chromosomes) showed a clear LET dependence for all energies. The ratio of simple to complex type exchanges increased with LET from 18 to 51%. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose response curve for chromosome damage with respect to gamma-rays. The RBE(sub max) values for total chromosome exchanges for the 64 MeV/u was around 30.

George, Kerry↗

The Affect of the Space Environment on the Survival of Halorubrum Chaoviator and Synechococcus (Nageli): Data from the Space Experiment OSMO on EXPOSE-R

We have shown using ESA's Biopan facility flown in Earth orbit that when exposed to the space environment for 2 weeks the survival rate of Synechococcus (Nageli), a halophilic cyanobacterium isolated from the evaporitic gypsum-halite crusts that form along the marine intertidal, and Halorubrum chaoviator a member of the Halobacteriaceae isolated from an evaporitic NaCl crystal obtained from a salt evaporation pond, were higher than all other test organisms except Bacillus spores. These results led to the EXPOSE-R mission to extend and refine these experiments as part of the experimental package for the external platform space exposure facility on the ISS. The experiment was flown in February 2009 and the organisms were exposed to low-Earth orbit for nearly 2 years. Samples were either exposed to solar ultraviolet (UV)-radiation (lambda is greater than 110 nm or lambda is greater than 200 nm, cosmic radiation (dosage range 225-320 mGy), or kept in darkness shielded from solar UV-radiation. Half of each of the UV-radiation exposed samples and dark samples were exposed to space vacuum and half kept at 105 pascals in argon. Duplicate samples were kept in the laboratory to serve as unexposed controls. Ground simulation control experiments were also performed. After retrieval, organism viability was tested using Molecular Probes Live-Dead Bac-Lite stain and by their reproduction capability. Samples kept in the dark, but exposed to space vacuum had a 90 +/- 5% survival rate compared to the ground controls. Samples exposed to full UV-radiation for over a year were bleached and although results from Molecular Probes Live-Dead stain suggested approximately 10% survival, the data indicate that no survival was detected using cell growth and division using the most probable number method. Those samples exposed to attenuated UV-radiation exhibited limited survival. Results from of this study are relevant to understanding adaptation and evolution of life, the future of life beyond earth, the potential for interplanetary transfer of viable microbes via meteorites and dust particles as well as spacecraft, and the physiology of halophiles.

space flight↗

Adherens junctions are involved in polarized contractile ring formation in dividing epithelial cells of Xenopus laevis embryos

Highlights: • The actomyosin contractile ring formation is polarized in epithelial cells. • Activation of the contractile ring is polarized in epithelial cells. • Adherens junctions are involved in the kinetics of the contractile ring formation. • Adherens junctions and the contractile ring are linked from the onset of cytokinesis. • High temporal resolution microscopy of epithelial cells in the Xenopus embryos. Cells dividing in the plane of epithelial tissues proceed by polarized constriction of the actomyosin contractile ring, leading to asymmetric ingression of the plasma mem brane. Asymmetric cytokinesis results in the apical positioning of the actomyosin contractile ring and ultimately of the midbody. Studies have indicated that the contractile ring is associated with adherens junctions, whose role is to maintain epithelial tissue cohesion. However, it is yet unknown when the contractile ring becomes associated with adherens junctions in epithelial cells. Here, we examined contractile ring formation and activation in the epithelium of Xenopus embryos and explored the implication of adherens junctions in the contractile ring formation. We show that accumulation of proteins involved in contractile ring formation and activation is polarized, starting at apical cell-cell contacts at the presumptive division site and spreading within seconds towards the cell basal side. We also show that adherens junctions are involved in the kinetics of contractile ring formation. Our study reveals that the link between the adherens junctions and the contractile ring is established from the onset of cytokinesis.

60 APPLIED LIFE SCIENCES↗

Luxury iron uptake and storage in pennate diatoms from the equatorial Pacific Ocean

Abstract Iron is a key micronutrient for ocean phytoplankton, and the availability of iron controls primary production and community composition in large regions of the ocean. Pennate diatoms, a phytoplankton group that responds to iron additions in low-iron areas, can have highly variable iron contents, and some groups such as Pseudo-nitzschia, are known to use ferritin to store iron for later use. We quantified and mapped the intracellular accumulation of iron by a natural population of Pseudo-nitzschia from the Fe-limited equatorial Pacific Ocean. A total of 48 h after iron addition, nearly half of the accumulated iron was localized in storage bodies adjacent to chloroplasts believed to represent ferritin. Over the subsequent 48 h, stored iron was distributed to the rest of the cell through subsequent growth and division, partially supporting the iron contents of the daughter cells. This study provides the first quantitative view into the cellular trafficking of iron in a globally relevant phytoplankton group and demonstrates the unique capabilities of synchrotron-based element imaging approaches.

59 BASIC BIOLOGICAL SCIENCES↗

Counting generations in birth and death processes with competing Erlang and exponential waiting times

Lymphocyte populations, stimulated in vitro or in vivo, grow as cells divide. Stochastic models are appropriate because some cells undergo multiple rounds of division, some die, and others of the same type in the same conditions do not divide at all. If individual cells behave independently, then each cell can be imagined as sampling from a probability density of times to division and death. The exponential density is the most mathematically and computationally convenient choice. It has the advantage of satisfying the memoryless property, consistent with a Markov process, but it overestimates the probability of short division times. With the aim of preserving the advantages of a Markovian framework while improving the representation of experimentally-observed division times, we consider a multi-stage model of cellular division and death. We use Erlang-distributed (or, more generally, phase-type distributed) times to division, and exponentially distributed times to death. We classify cells into generations, using the rule that the daughters of cells in generation n are in generation n + 1. In some circumstances, our representation is equivalent to established models of lymphocyte dynamics. We find the growth rate of the cell population by calculating the proportions of cells by stage and generation. The exponent describing the late-time cell population growth, and the criterion for extinction of the population, differs from what would be expected if N steps with rate λ were equivalent to a single step of rate λ/N. We link with a published experimental dataset, where cell counts were reported after T cells were transferred to lymphopenic mice, using Approximate Bayesian Computation. In the comparison, the death rate is assumed to be proportional to the generation and the Erlang time to division for generation 0 is allowed to differ from that of subsequent generations. The multi-stage representation is preferred to a simple exponential in posterior distributions, and the mean time to first division is estimated to be longer than the mean time to subsequent divisions.

59 BASIC BIOLOGICAL SCIENCES↗

Chromosomal Aberrations in DNA Repair Defective Cell Lines: Comparisons of Dose Rate and Radiation Quality

Chromosome aberration yields were assessed in DNA double-strand break repair (DSB) deficient cells after acute doses of gamma-rays or high-LET iron nuclei, or low dose-rate (0.018 Gy/hr) gamma-rays. We studied several cell lines including fibroblasts deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase, DNA-PK activity. Chromosomes were analyzed using the fluorescence in-situ hybridization (FISH) chromosome painting method in cells at the first division post-irradiation and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma radiation induced higher yields of both simple and complex exchanges in the DSB repair defective cells than in the normal cells. The quadratic dose-response terms for both chromosome exchange types were significantly higher for the ATM and NBS defective lines than for normal fibroblasts. However, the linear dose-response term was significantly higher only for simple exchanges in the NBS cells. Large increases in the quadratic dose response terms indicate the important roles of ATM and NBS in chromatin modifications that facilitate correct DSB repair and minimize aberration formation. Differences in the response of AT and NBS deficient cells at lower doses suggests important questions about the applicability of observations of radiation sensitivity at high dose to low dose exposures. For all iron nuclei irradiated cells, regression models preferred purely linear and quadratic dose responses for simple and complex exchanges, respectively. All the DNA repair defective cell lines had lower Relative biological effectiveness (RBE) values than normal cells, the lowest being for the DNA-PK-deficient cells, which was near unity. To further investigate the sensitivity differences for low and low high doses, we performed chronic low dose-rate irradiation, and have begun studies with ATM and Nibrin inhibitors and siRNA knockout of these proteins. Results support the conclusion that for the endpoint of simple chromosomal aberrations (translocation or dicentrics), the increased radiation sensitivity of AT cells found at high doses (>1 Gy) does not carry over to low doses or doserates, while NBS cells show increased sensitivity for both high and low dose exposures.

George, K. A.↗

DNA Repair Defects and Chromosomal Aberrations

Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of the DNA repair-defective cell lines were smaller than those of normal cells, with the DNA-PK-deficient cells having RBEs near unity. To further investigate the sensitivity differences that were observed in ATM and NBS deficient cells, chromosomal aberrations were analyzed in normal lung fibroblast cells treated with KU-55933 (a specific ATM kinase inhibitor) or Mirin (an Mre11- Rad50-Nbs1 complex inhibitor involved in activation of ATM). We also performed siRNA knockdown of these proteins. Preliminary data indicate that chromosome exchanges increase in cells treated with the specific ATM inhibitor. Possible cytogenetic signatures of acute and low dose-rate gamma irradiation in ATM or nibrin deficient and suppressed cells will be discussed.

Hada, Megumi↗

Systems Analysis Initiated for All-Electric Aircraft Propulsion

A multidisciplinary effort is underway at the NASA Glenn Research Center to develop concepts for revolutionary, nontraditional fuel cell power and propulsion systems for aircraft applications. There is a growing interest in the use of fuel cells as a power source for electric propulsion as well as an auxiliary power unit to substantially reduce or eliminate environmentally harmful emissions. A systems analysis effort was initiated to assess potential concepts in an effort to identify those configurations with the highest payoff potential. Among the technologies under consideration are advanced proton exchange membrane (PEM) and solid oxide fuel cells, alternative fuels and fuel processing, and fuel storage. Prior to this effort, the majority of fuel cell analysis done at Glenn was done for space applications. Because of this, a new suite of models was developed. These models include the hydrogen-air PEM fuel cell; internal reforming solid oxide fuel cell; balance-of-plant components (compressor, humidifier, separator, and heat exchangers); compressed gas, cryogenic, and liquid fuel storage tanks; and gas turbine/generator models for hybrid system applications. Initial mass, volume, and performance estimates of a variety of PEM systems operating on hydrogen and reformate have been completed for a baseline general aviation aircraft. Solid oxide/turbine hybrid systems are being analyzed. In conjunction with the analysis efforts, a joint effort has been initiated with Glenn s Computer Services Division to integrate fuel cell stack and component models with the visualization environment that supports the GRUVE lab, Glenn s virtual reality facility. The objective of this work is to provide an environment to assist engineers in the integration of fuel cell propulsion systems into aircraft and provide a better understanding of the interaction between system components and the resulting effect on the overall design and performance of the aircraft. Initially, three-dimensional computer-aided design (CAD) models of representative PEM fuel cell stack and components were developed and integrated into the virtual reality environment along with an Excel-based model used to calculate fuel cell electrical performance on the basis of cell dimensions (see the figure). CAD models of a representative general aviation aircraft were also developed and added to the environment. With the use of special headgear, users will be able to virtually manipulate the fuel cell s physical characteristics and its placement within the aircraft while receiving information on the resultant fuel cell output power and performance. As the systems analysis effort progresses, we will add more component models to the GRUVE environment to help us more fully understand the effect of various system configurations on the aircraft.

Kohout, Lisa L.↗

Study of radiation effects on mammalian cells in vitro

Radiation effect on single cells and cell populations of Chinese hamster lung tissue is studied in vitro. The rate and position as the cell progresses through the generation cycle shows division delay, changes in some biochemical processes in the cell, chromosomal changes, colony size changes, and loss of reproductive capacity.

Sinclair, W. K.↗