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Chromatin instability and protein homeostasis (proteostasis) stress are two well-established hallmarks of aging, which have been considered largely independent of each other. Using microfluidics and single-cell imaging approaches, we observed that, during the replicative aging of Saccharomyces cerevisiae, a challenge to proteostasis occurs specifically in the fraction of cells with decreased stability within the ribosomal DNA (rDNA). A screen of 170 yeast RNA-binding proteins identified ribosomal RNA (rRNA)-binding proteins as the most enriched group that aggregate upon a decrease in rDNA stability induced by inhibition of a conserved lysine deacetylase Sir2. Further, loss of rDNA stability induces age-dependent aggregation of rRNA-binding proteins through aberrant overproduction of rRNAs. These aggregates contribute to age-induced proteostasis decline and limit cellular lifespan. Our findings reveal a mechanism underlying the interconnection between chromatin instability and proteostasis stress and highlight the importance of cell-to-cell variability in aging processes.

The emerging technology of brain organoids deriving from human pluripotent stem cells provides unprecedented opportunities to study human brain development and associated disorders. Various brain organoid protocols have been developed that can recapitulate some key features of cell type diversity, cytoarchitectural organization, developmental processes, functions, and pathologies of the developing human brain. In this review, we focus on patterning of human stem cell-derived brain organoids. We start with an overview of general procedures to generate brain organoids. We then highlight some recently developed brain organoid protocols and chemical cues involved in modeling development of specific human brain regions, subregions, and multiple regions together. We also discuss limitations and potential future improvements of human brain organoid technology.

Aging is a complex, yet pervasive phenomenon in biology. As human cells steadily succumb to the deteriorating effects of aging, so too comes a host of age-related ailments such as neurodegenerative disorders, cardiovascular disease and cancer. Therefore, elucidation of the molecular networks that drive aging is of paramount importance to human health. Progress toward this goal has been aided by studies from simple model organisms such as Saccharomyces cerevisiae. While work in budding yeast has already revealed much about the basic biology of aging as well as a number of evolutionarily conserved pathways involved in this process, recent technological advances are poised to greatly expand our knowledge of aging in this simple eukaryote. Here, we review the latest developments in microfluidics, single-cell analysis and high-throughput technologies for studying single-cell replicative aging in S. cerevisiae. We detail the challenges each of these methods addresses as well as the unique insights into aging that each has provided. We conclude with a discussion of potential future applications of these techniques as well as the importance of single-cell dynamics and quantitative biology approaches for understanding cell aging.

SummaryChromatin instability and loss of protein homeostasis (proteostasis) are two well-established hallmarks of aging, which have been considered largely independent of each other. Using microfluidics and single-cell imaging approaches, we observed that, during the replicative aging of S.cerevisiae, proteostasis decline occurred specifically in the fraction of cells with decreased stability at the ribosomal DNA (rDNA) region. A screen of 170 yeast RNA-binding proteins identified ribosomal RNA (rRNA)- binding proteins as the most enriched group that aggregate upon a decrease in rDNA stability. We further found that loss of rDNA stability contributes to age-dependent aggregation of rRNA-binding proteins through aberrant overproduction of rRNAs. These aggregates negatively impact nucleolar integrity and global proteostasis and hence limit cellular lifespan. Our findings reveal a mechanism underlying the interconnection between chromatin instability and proteostasis decline and highlight the importance of cell-to-cell variability in aging processes.

Aging is a complex, yet pervasive phenomenon in biology. As human cells steadily succumb to the deteriorating effects of aging, so too comes a host of age-related ailments such as neurodegenerative disorders, cardiovascular disease and cancer. Therefore, elucidation of the molecular networks that drive aging is of paramount importance to human health. Progress toward this goal has been aided by studies from simple model organisms such as Saccharomyces cerevisiae. While work in budding yeast has already revealed much about the basic biology of aging as well as a number of evolutionarily conserved pathways involved in this process, recent technological advances are poised to greatly expand our knowledge of aging in this simple eukaryote. Here, we review the latest developments in microfluidics, single-cell analysis and high-throughput technologies for studying single-cell replicative aging in S. cerevisiae. We detail the challenges each of these methods addresses as well as the unique insights into aging that each has provided. We conclude with a discussion of potential future applications of these techniques as well as the importance of single-cell dynamics and quantitative biology approaches for understanding cell aging.

Programmed aging in yeast cells Following the fate of individual yeast cells has revealed aging to be more of a programmable decision process rather than a simple accumulation of deleterious events. Li et al. combined single-cell studies and mathematical modeling to show that yeast cells showed two different forms of aging: one with less ribosomal DNA silencing, in which nucleoli were degraded, and another with less heme accumulation and hemedependent transcription, in which mitochondria were more affected. Overexpression of the lysine deacetylase Sir2, which contributes to ribosomal DNA silencing, led to a third cell-aging fate in which the average life span was extended. If other cells age in similar ways, then this study may provide new ways to consider dynamics of aging and strategies to extend the health span. Science this issue p. 325

Chromatin instability and mitochondrial decline are conserved processes that contribute to cellular aging. Although both processes have been explored individually in the context of their distinct signaling pathways, the mechanism that determines which cell fate arises in isogenic cells is unknown. Here, we show that interactions between the chromatin silencing and mitochondrial pathways lead to an epigenetic landscape with multiple equilibrium states that represent different types of terminal cellular states. Interestingly, the structure of the landscape drives single-cell differentiation towards one of these states during aging, whereby the fate is determined quite early and is insensitive to intracellular noise. Guided by a quantitative model of the aging landscape, we genetically engineer a new “long-lived” equilibrium state that is characterized by a dramatically extended lifespan.

Cellular aging is a complex process that involves many interwoven molecular processes. Studies in model organisms have identified many individual genes and factors that have profound effects on lifespan. However, how these genes and factors interact and function collectively to drive the aging process remains unclear. We investigated single-cell aging dynamics throughout the replicative lifespans of S. cerevisiae, and found that isogenic cells diverge towards two aging paths, with distinct phenotypic changes and death forms. We further identified specific molecular pathways driving each aging fate and revealed that these pathways interact and operate dynamically to enable an early-life switch that governs the aging fate decision and the progression towards death. Our work uncovers the interconnected molecular pathways that drives the aging process and opens up the possibility of designing interventions that simultaneously target multiple network nodes, instead of single genes, to more effectively extend the healthspan.

Cellular aging plays an important role in many diseases, such as cancers, metabolic syndromes, and neurodegenerative disorders. There has been steady progress in identifying aging-related factors such as reactive oxygen species and genomic instability, yet an emerging challenge is to reconcile the contributions of these factors with the fact that genetically identical cells can age at significantly different rates. Such complexity requires single-cell analyses designed to unravel the interplay of aging dynamics and cell-to-cell variability. Here we use microfluidic technologies to track the replicative aging of single yeast cells and reveal that the temporal patterns of heterochromatin silencing loss regulate cellular life span. We found that cells show sporadic waves of silencing loss in the heterochromatic ribosomal DNA during the early phases of aging, followed by sustained loss of silencing preceding cell death. Isogenic cells have different lengths of the early intermittent silencing phase that largely determine their final life spans. Combining computational modeling and experimental approaches, we found that the intermittent silencing dynamics is important for longevity and is dependent on the conserved Sir2 deacetylase, whereas either sustained silencing or sustained loss of silencing shortens life span. These findings reveal that the temporal patterns of a key molecular process can directly influence cellular aging, and thus could provide guidance for the design of temporally controlled strategies to extend life span.

The yeast metabolic cycle (YMC) is a fascinating example of biological organization, in which cells constrain the function of specific genetic, protein and metabolic networks to precise temporal windows as they grow and divide. However, understanding the intracellular origins of the YMC remains a challenging goal, as measuring the oxygen oscillations traditionally associated with it requires the use of synchronized cultures growing in nutrient-limited chemostat environments. To address these limitations, we used custom-built microfluidic devices and time-lapse fluorescence microscopy to search for metabolic cycling in the form of endogenous flavin fluorescence in unsynchronized single yeast cells. We uncovered robust and pervasive metabolic cycles that were synchronized with the cell division cycle (CDC) and oscillated across four different nutrient conditions. We then studied the response of these metabolic cycles to chemical and genetic perturbations, showing that their phase synchronization with the CDC can be altered through treatment with rapamycin, and that metabolic cycles continue even in respiratory deficient strains. These results provide a foundation for future studies of the physiological importance of metabolic cycles in processes such as CDC control, metabolic regulation and cell aging.

Cellular aging plays an important role in many diseases, such as cancers, metabolic syndromes and neurodegenerative disorders. There has been steady progress in identifying aging-related factors such as reactive oxygen species and genomic instability, yet an emerging challenge is to reconcile the contributions of these factors with the fact that genetically identical cells can age at significantly different rates. Such complexity requires single-cell analyses designed to unravel the interplay of aging dynamics and cell-to-cell variability. Here we use novel microfluidic technologies to track the replicative aging of single yeast cells and reveal that the temporal patterns of heterochromatin silencing loss regulate cellular lifespan. We found that cells show sporadic waves of silencing loss in the heterochromatic ribosomal DNA (rDNA) during the early phases of aging, followed by sustained loss of silencing preceding cell death. Isogenic cells have different lengths of the early intermittent silencing phase that largely determine their final lifespans. Combining computational modeling and experimental approaches, we found that the intermittent silencing dynamics is important for longevity and is dependent on the conserved Sir2 deacetylase, whereas either sustained silencing or sustained loss of silencing shortens lifespan. These findings reveal, for the first time, that the temporal patterns of a key molecular process can directly influence cellular aging and thus could provide guidance for the design of temporally controlled strategies to extend lifespan.SignificanceAging is an inevitable consequence of living, and with it comes increased morbidity and mortality. Novel approaches to mitigating age-related chronic diseases demand a better understanding of the biology of aging. Studies in model organisms have identified many conserved molecular factors that influence aging. The emerging challenge is to understand how these factors interact and change dynamically to drive aging. Using multidisciplinary technologies, we have revealed a sirtuin-dependent intermittent pattern of chromatin silencing during yeast aging that is crucial for longevity. Our findings highlight the important role of silencing dynamics in aging, which deserves careful consideration when designing schemes to delay or reverse aging by modulating sirtuins and silencing.

Summary Although genetic mutations that alter organisms’ average lifespans have been identified in aging research, our understanding of the dynamic changes during aging remains limited. Here, we integrate single-cell imaging, microfluidics, and computational modeling to investigate phenotypic divergence and cellular heterogeneity during replicative aging of single S. cerevisiae cells. Specifically, we find that isogenic cells diverge early in life toward one of two aging paths, which are characterized by distinct age-associated phenotypes. We captured the dynamics of single cells along the paths with a stochastic discrete-state model, which accurately predicts both the measured heterogeneity and the lifespan of cells on each path within a cell population. Our analysis suggests that genetic and environmental factors influence both a cell’s choice of paths and the kinetics of paths themselves. Given that these factors are highly conserved throughout eukaryotes, divergent aging might represent a general scheme in cellular aging of other organisms.

One of the major reasons of failure in Total Knee Arthroplasty (TKA) is aseptic loosening of the tibial implant. Currently, cement viscosity’s role in ensuring the long-term success of the arthroplasty is not clear. We conducted the failure analysis of the surrogate tibial constructs instrumented with fast and medium setting bone cements. The three cements (Depuy 2, Palacos R (fast setting, high viscosity cements) and Simplex P, (medium setting, a low viscosity cement)) were used to prepare the tibial constructs using standard TKA procedures. Mechanical load tests were conducted to investigate the structural integrity of the constructs. Average cement penetration was measured from radiographs of the constructs and analyzed according to the Knee Society Total Knee Arthroplasty Roentgenographic Evaluation System. Simplex P had the maximum cumulative penetration in seven zones in the mediolateral view, and three zones in antero-posterior view. Simplex P exhibited maximum penetration in zone 7 in the anteroposterior view, and in zone 3 in the lateral view. For the mechanical tests the TKA constructs were subjected to cyclic compressive loading (600 N, 5000 cycles at 10 Hz) in the sagittal plane. Simplex P displayed the smallest micromotion of the three cements. Displacement of the tibial trays with Simplex P, 0.728 ± 0.156 mm, and Palacos R, 1.573 ± 0.519 mm had no statistically significant difference ( P = 0.109). However, the tibial trays with Simplex P showed statistically significant smaller displacement than those with Depuy 2, 2.267 ± 0.491 mm ( P = 0.010). There was no significant difference between the displacements for Palacos R and Depuy 2 ( P = 0.190). Digital Imaging Correlation (DIC) was used to determine displacements in the transverse plane. In the transverse plane, Simplex P had the lowest micromotion, however the results were not significant ( P > 0.05). The consistently superior performance of Simplex P suggests that cement viscosity does indeed play a role in arthroplasty success. These results have direct clinical relevance for TKA patients suffering from aseptic loosening.

Aseptic loosening of the tibial implant remains one of the major reasons of failure in Total Knee Arthroplasty (TKA). Currently, there is no consensus on the role that cement viscosity at the time of application to the bone plays in ensuring the long-term success of the arthroplasty. The purpose of this study was to investigate the relationship between cement viscosity and aseptic loosening of tibial implants. Three cements (Depuy 2, Palacos R (high viscosity cements) and Simplex-P, a medium viscosity cement) were compared during TKA through radiographic analysis and mechanical loading tests using surrogate tibia. Cement penetration was measured from radiographs of the constructs and analyzed according to the Knee Society Total Knee Arthroplasty Roentgenographic Evaluation System. Simplex-P had the maximum cumulative penetration in seven zones in the mediolateral view, and three zones in antero-posterior view. Simplex exhibited maximum penetration in zone 7 in the antero-posterior view, and in zone 3 in the lateral view. For the mechanical tests the TKA constructs were subjected to cyclic compressive loading in the sagittal plane. Simplex-P had the smallest micro-motion in sagittal plane, the results were significant when compared to Palacos R. The consistently superior performance of Simplex-P suggests that cement viscosity does indeed play a role in arthroplasty success. These results have direct clinical relevance for TKA patients suffering from aseptic loosening.Copyright © 2012 by ASME

Premature infants make up 25% of all births in developing countries and are usually born outside of a hospital. The World Health Organization and Engineering World Health have stressed the need for a transportable infant incubator designed for use in developing countries. The goal of this project is to create a device which protects and incubates an infant while being transported to a hospital. The device needs to be economical, robust, and use easily replaceable parts. The heating mechanism will use computer fans, a humidifier, and car headlights to provide heat. The temperature will be automatically maintained by a proportional integrative derivative (PID) controller. Large temperature deviations and low battery charge will be announced through an audible alert. The base will be made of aluminum due to its low cost and durability. The bassinet cover will be polycarbonate due to its low thermal conductivity and strength. A theoretical MATLAB model of the preterm infant and incubator will be implemented to evaluate the performance of the design. Testing goals will include finding average power consumption, times to reach target temperature (33.5°C), and the safety of the device.