Fluorometric and colorimetric sensor for selective detection of cyanide anion by dibenzosuberenone-based dihydropyridazine in aqueous solution.

A new chemosensory based on deprotonation and intramolecular charge transfer (ICT) was developed to detect cyanide in food samples. Deprotonation was facilitated by increasing the acidity of the NH proton in the dibenzosuberenone-based dihydropyridazine chemosensor Pz3 with -CN substituents. Addition of cyanide to acetonitrile and aqueous acetonitrile solution (1/9) of Pz3 resulted in their significant color change from colorless to purple in visible light, accompanied by a strong red shift in the absorption spectrum. Meanwhile, the near-infrared (NIR) emission (ex. 525 nm, em. 670 nm) of Pz3- resulting from deprotonation showed fluorescence switching behavior to detect the cyanide anion. While the acidic NH protons interact with basic anions as F-, CN-, OAc- and H2PO4- in organic solution (MeCN), just CN ions interact with in aqueous organic solutions (H2O-MeCN 1/9 HEPES pH 7.4). The limit of detection of cyanide from the fluorescence spectrum is 80 nM, which is well below the value determined for drinking water by World Health Organization (WHO). The interference effect of cations and anions showed that Pz3 could play an important role in the determination of waste NaCN. In addition, Pz3 successfully carried out the selective detection of cyanide in food samples such as bitter almonds and sprouting potatoes.

Astaxanthin and meclizine extend lifespan in UM-HET3 male mice; fisetin, SG1002 (hydrogen sulfide donor), dimethyl fumarate, mycophenolic acid, and 4-phenylbutyrate do not significantly affect lifespan in either sex at the doses and schedules used.

In genetically heterogeneous (UM-HET3) mice produced by the CByB6F1 × C3D2F1 cross, the Nrf2 activator astaxanthin (Asta) extended the median male lifespan by 12% (p = 0.003, log-rank test), while meclizine (Mec), an mTORC1 inhibitor, extended the male lifespan by 8% (p = 0.03). Asta was fed at 1840 ± 520 (9) ppm and Mec at 544 ± 48 (9) ppm, stated as mean ± SE (n) of independent diet preparations. Both were started at 12 months of age. The 90th percentile lifespan for both treatments was extended in absolute value by 6% in males, but neither was significant by the Wang-Allison test. Five other new agents were also tested as follows: fisetin, SG1002 (hydrogen sulfide donor), dimethyl fumarate, mycophenolic acid, and 4-phenylbutyrate. None of these increased lifespan significantly at the dose and method of administration tested in either sex. Amounts of dimethyl fumarate in the diet averaged 35% of the target dose, which may explain the absence of lifespan effects. Body weight was not significantly affected in males by any of the test agents. Late life weights were lower in females fed Asta and Mec, but lifespan was not significantly affected in these females. The male-specific lifespan benefits from Asta and Mec may provide insights into sex-specific aspects of aging.

Sis2 regulates yeast replicative lifespan in a dose-dependent manner.

Application of microfluidic platforms facilitated high-precision measurements of yeast replicative lifespan (RLS); however, comparative quantification of lifespan across strain libraries has been missing. Here we microfluidically measure the RLS of 307 yeast strains, each deleted for a single gene. Despite previous reports of extended lifespan in these strains, we found that 56% of them did not actually live longer than the wild-type; while the remaining 44% showed extended lifespans, the degree of extension was often different from what was previously reported. Deletion of SIS2 gene led to the largest RLS increase observed. Sis2 regulated yeast lifespan in a dose-dependent manner, implying a role for the coenzyme A biosynthesis pathway in lifespan regulation. Introduction of the human PPCDC gene in the sis2Δ background neutralized the lifespan extension. RNA-seq experiments revealed transcriptional increases in cell-cycle machinery components in sis2Δ background. High-precision lifespan measurement will be essential to elucidate the gene network governing lifespan.

Phenotypic plasticity as a facilitator of microbial evolution.

Tossed about by the tides of history, the inheritance of acquired characteristics has found a safe harbor at last in the rapidly expanding field of epigenetics. The slow pace of genetic variation and high opportunity cost associated with maintaining a diverse genetic pool are well-matched by the flexibility of epigenetic traits, which can enable low-cost exploration of phenotypic space and reactive tuning to environmental pressures. Aiding in the generation of a phenotypically plastic population, epigenetic mechanisms often provide a hotbed of innovation for countering environmental pressures, while the potential for genetic fixation can lead to strong epigenetic-genetic evolutionary synergy. At the level of cells and cellular populations, we begin this review by exploring the breadth of mechanisms for the storage and intergenerational transmission of epigenetic information, followed by a brief review of common and exotic epigenetically regulated phenotypes. We conclude by offering an in-depth coverage of recent papers centered around two critical issues: the evolvability of epigenetic traits through Baldwinian adaptive phenotypic plasticity and the potential for synergy between epigenetic and genetic evolution.

Phenotypic selection during laboratory evolution of yeast populations leads to a genome-wide sustainable chromatin compaction shift.

In a previous study, we have shown how microbial evolution has resulted in a persistent reduction in expression after repeatedly selecting for the lowest PGAL1-YFP-expressing cells. Applying the ATAC-seq assay on samples collected from this 28-day evolution experiment, here we show how genome-wide chromatin compaction changes during evolution under selection pressure. We found that the chromatin compaction was altered not only on GAL network genes directly impacted by the selection pressure, showing an example of selection-induced non-genetic memory, but also at the whole-genome level. The GAL network genes experienced chromatin compaction accompanying the reduction in PGAL1-YFP reporter expression. Strikingly, the fraction of global genes with differentially compacted chromatin states accounted for about a quarter of the total genome. Moreover, some of the ATAC-seq peaks followed well-defined temporal dynamics. Comparing peak intensity changes on consecutive days, we found most of the differential compaction to occur between days 0 and 3 when the selection pressure was first applied, and between days 7 and 10 when the pressure was lifted. Among the gene sets enriched for the differential compaction events, some had increased chromatin availability once selection pressure was applied and decreased availability after the pressure was lifted (or vice versa). These results intriguingly show that, despite the lack of targeted selection, transcriptional availability of a large fraction of the genome changes in a very diverse manner during evolution, and these changes can occur in a relatively short number of generations.

Modeling aging and its impact on cellular function and organismal behavior.

Aging is a complex phenomenon of functional decay in a biological organism. Although the effects of aging are readily recognizable in a wide range of organisms, the cause(s) of aging are ill defined and poorly understood. Experimental methods on model organisms have driven significant insight into aging as a process, but have not provided a complete model of aging. Computational biology offers a unique opportunity to resolve this gap in our knowledge by generating extensive and testable models that can help us understand the fundamental nature of aging, identify the presence and characteristics of unaccounted aging factor(s), demonstrate the mechanics of particular factor(s) in driving aging, and understand the secondary effects of aging on biological function. In this review, we will address each of the above roles for computational biology in aging research. Concurrently, we will explore the different applications of computational biology to aging in single-celled versus multicellular organisms. Given the long history of computational biogerontological research on lower eukaryotes, we emphasize the key future goals of gradually integrating prior models into a holistic map of aging and translating successful models to higher-complexity organisms.

A yeast-optimized single-cell transcriptomics platform elucidates how mycophenolic acid and guanine alter global mRNA levels.

Stochastic gene expression leads to inherent variability in expression outcomes even in isogenic single-celled organisms grown in the same environment. The Drop-Seq technology facilitates transcriptomic studies of individual mammalian cells, and it has had transformative effects on the characterization of cell identity and function based on single-cell transcript counts. However, application of this technology to organisms with different cell size and morphology characteristics has been challenging. Here we present yeastDrop-Seq, a yeast-optimized platform for quantifying the number of distinct mRNA molecules in a cell-specific manner in individual yeast cells. Using yeastDrop-Seq, we measured the transcriptomic impact of the lifespan-extending compound mycophenolic acid and its epistatic agent guanine. Each treatment condition had a distinct transcriptomic footprint on isogenic yeast cells as indicated by distinct clustering with clear separations among the different groups. The yeastDrop-Seq platform facilitates transcriptomic profiling of yeast cells for basic science and biotechnology applications.

Chromatin regulatory genes differentially interact in networks to facilitate distinct GAL1 activity and noise profiles.

Controlling chromatin state constitutes a major regulatory step in gene expression regulation across eukaryotes. While global cellular features or processes are naturally impacted by chromatin state alterations, little is known about how chromatin regulatory genes interact in networks to dictate downstream phenotypes. Using the activity of the canonical galactose network in yeast as a model, here, we measured the impact of the disruption of key chromatin regulatory genes on downstream gene expression, genetic noise and fitness. Using Trichostatin A and nicotinamide, we characterized how drug-based modulation of global histone deacetylase activity affected these phenotypes. Performing epistasis analysis, we discovered phenotype-specific genetic interaction networks of chromatin regulators. Our work provides comprehensive insights into how the galactose network activity is affected by protein interaction networks formed by chromatin regulators.

Epigenetic Mechanisms Contribute to Evolutionary Adaptation of Gene Network Activity under Environmental Selection.

How evolution can be facilitated by epigenetic mechanisms has received refreshed attention recently. To explore the role epigenetic inheritance plays in evolution, we subject isogenic wild-type yeast cells expressing PGAL1-YFP (yellow fluorescent protein) to selection by daily sorting based on reporter expression. We observe expression-level reductions in multiple replicates sorted for the lowest expression that persist for several days, even after lifting the selection pressure. Reduced expression is due to factors in the galactose (GAL) network rather than global factors. Results using a constitutively active GAL network are in overall agreement with findings with the wild-type network. We find that the local chromatin environment of the reporter has a significant effect on the observed phenotype. Genome sequencing, chromatin immunoprecipitation (ChIP)-qPCR, and sporulation analysis provide further insights into the epigenetic and genetic contributors to the expression changes observed. Our work provides a comprehensive example of the role played by epigenetic mechanisms on gene network evolution.

Photophysical Properties of Some Fluorescent Dyes in SDS Micellar Solutions.

Photophysical properties of fluorescent dyes such as Safranin T, Acridine Orange, Pyronin B and Pyronin Y in SDS micelles were examined by using spectroscopic techniques. Firstly, spherical micelles in deionized water were prepared with Sodium Dodecyl Sulfate (SDS) surfactants and they were transformed into their layered structures (lamellar micelles) by the aid of NaCl (sodium chloride). SEM studies confirmed the transformation of SDS micelles from the spherical structures to the lamellar structures. Secondly, absorption and fluorescence characteristics of the dyes in deionized water and the SDS micelles aqueous solutions were characterized in the presence of various NaCl concentrations at above the critical micelle concentration (CMC). Moreover, the photophysical properties of the dyes in various media were discussed by fluorescence quantum yield and fluorescence lifetime data. The micellar structures called a mimetic membrane system changed the photophysical properties of the dyes compared to those in deionized water.

Characterization of the impact of GMP/GDP synthesis inhibition on replicative lifespan extension in yeast.

Slowing down aging-associated accumulation of molecular damage or its prevention represents a promising therapeutic paradigm to combat aging-related disease and death. While several chemical compounds extend lifespan in model organisms, their mechanism of action is often unknown, reducing their therapeutic potential. Using a systematic approach, here we characterize the impact of the GMP pathway on yeast lifespan and elucidate GMP synthesis inhibition as a lifespan extension mechanism. We further discover that proteasome activation extends lifespan in part through the GMP pathway. GMP synthesis inhibition exerts its lifespan extension effect independently of the canonical nutrient-sensing pathway regulating lifespan. Exposing longitudinally aging yeast cells to GMP pathway inhibition in an age-dependent manner, we demonstrate that the lifespan extension is facilitated by slowing, rather than reversing, the aging process in cells. Using a GUK1 mutant with lower GMP-to-GDP conversion activity, we observe lifespan extension, suggesting that reduced GDP level by itself can also extend yeast lifespan. These findings elucidate the involvement of nucleotide metabolism in the aging process. The existence of clinically-approved GMP pathway inhibitors elicits the potential of a new class of therapeutics for aging-related disorders.

Quantitative Insights into Age-Associated DNA-Repair Inefficiency in Single Cells.

Although double-strand break (DSB) repair is essential for a cell's survival, little is known about how DSB repair mechanisms are affected by age. Here we characterize the impact of cellular aging on the efficiency of single-strand annealing (SSA), a DSB repair mechanism. We measure SSA repair efficiency in young and old yeast cells and report a 23.4% decline in repair efficiency. This decline is not due to increased use of non-homologous end joining. Instead, we identify increased G1 phase duration in old cells as a factor responsible for the decreased SSA repair efficiency. Expression of 3xCLN2 leads to higher SSA repair efficiency in old cells compared with expression of 1xCLN2, confirming the involvement of cell-cycle regulation in age-associated repair inefficiency. Examining how SSA repair efficiency is affected by sequence heterology, we find that heteroduplex rejection remains high in old cells. Our work provides insights into the links between single-cell aging and DSB repair efficiency.

Stochastic modeling of aging cells reveals how damage accumulation, repair, and cell-division asymmetry affect clonal senescence and population fitness.

BACKGROUND: Asymmetry during cellular division, both in the uneven partitioning of damaged cellular components and of cell volume, is a cell biological phenomenon experienced by many unicellular organisms. Previous work based on a deterministic model claimed that such asymmetry in the partitioning of cell volume and of aging-associated damage confers a fitness benefit in avoiding clonal senescence, primarily by diversifying the cellular population. However, clonal populations of unicellular organisms are already naturally diversified due to the inherent stochasticity of biological processes. RESULTS: Applying a model of aging cells that accounts for natural cell-to-cell variations across a broad range of parameter values, here we show that the parameters directly controlling the accumulation and repair of damage are the most important factors affecting fitness and clonal senescence, while the effects of both segregation of damaged components and division asymmetry are frequently minimal and generally context-dependent. CONCLUSIONS: We conclude that damage segregation and division asymmetry, perhaps counterintuitively, are not necessarily beneficial from an evolutionary perspective.

Insights into Bidirectional Gene Expression Control Using the Canonical GAL1/GAL10 Promoter.

Despite advances made in understanding the effects of promoter structure on transcriptional activity, limited knowledge exists regarding the role played by chromatin architecture in transcription. Previous work hypothesized that transcription from the bidirectional GAL1/GAL10 promoter is controlled through looping of its UAS region around a nonstandard nucleosome. Here, by editing the GAL1/GAL10 promoter at high resolution, we provide insights into bidirectional expression control. We demonstrate that the first and fourth Gal4 binding sites within the UAS do not functionally contribute to promoter activation. Instead, these sites, along with nearby regulatory regions, contribute to the directional regulation of gene expression. Furthermore, Gal4 binding to the third binding site is critical for gene expression, while binding to the other three sites is not sufficient for transcriptional activation. Because the GAL1/GAL10 UAS can activate gene expression in many eukaryotes, the regulatory mechanism presented is expected to operate broadly across the eukaryotic clade.

Multi-component gene network design as a survival strategy in diverse environments.

BACKGROUND: Gene-environment interactions are often mediated though gene networks in which gene expression products interact with other network components to dictate network activity levels, which in turn determines the fitness of the host cell in specific environments. Even though a gene network is the right context for studying gene-environment interactions, we have little understanding on how systematic genetic perturbations affects fitness in the context of a gene network. RESULTS: Here we examine the effect of combinatorial gene dosage alterations on gene network activity and cellular fitness. Using the galactose utilization pathway as a model network in diploid yeast, we reduce the copy number of four regulatory genes (GAL2, GAL3, GAL4, GAL80) from two to one, and measure the activity of the perturbed networks. We integrate these results with competitive fitness measurements made in six different rationally-designed environments containing different galactose concentrations representing the natural induction spectrum of the galactose network. In the lowest galactose environment, we find a nonlinear relationship between gene expression and fitness while high galactose environments lead to a linear relationship between the two with a saturation regime reached at a sufficiently high galactose concentration. We further uncover environment-specific relevance of the different network components for dictating the relationship between the network activity and organismal fitness, indicating that none of the network components are redundant. CONCLUSIONS: These results provide experimental support to the hypothesis that dynamic changes in the environment throughout natural evolution is key to structuring natural gene networks in a multi-component fashion, which robustly provides protection against population extinction in different environments.

The Systems Biology of Single-Cell Aging.

Aging is a leading cause of human morbidity and mortality, but efforts to slow or reverse its effects are hampered by an incomplete understanding of its multi-faceted origins. Systems biology, the use of quantitative and computational methods to understand complex biological systems, offers a toolkit well suited to elucidating the root cause of aging. We describe the known components of the aging network and outline innovative techniques that open new avenues of investigation to the aging research community. We propose integration of the systems biology and aging fields, identifying areas of complementarity based on existing and impending technological capabilities.

Fundamental Characteristics of Single-Cell Aging in Diploid Yeast.

Single-cell-level experimentation can elucidate key biological insights about cellular aging that are masked in population-level studies. However, the extensive time requirement of tracking single cells has historically prevented their long-term longitudinal observation. Using a microfluidic device that automates microscopic monitoring of diploid Saccharomyces cerevisiae cells throughout their replicative lifespan, here we report the fundamental characteristics of single-cell aging for diploid yeast. We find that proteins with short versus long half-lives exhibit distinct dynamics as cells age and that the intercellular gene expression noise increases during aging, whereas the intracellular noise stays unchanged. A stochastic model provides quantitative mechanistic insights into the observed noise dynamics and sheds light on the age-dependent intracellular noise differences between diploid and haploid yeast. Our work elucidates how a set of canonical phenotypes dynamically change while the host cells are aging in real time, providing essential insights for a comprehensive understanding on and control of lifespan at the single-cell level.

Live-Cell Imaging of Chromatin Condensation Dynamics by CRISPR.

The spatiotemporal organization of chromatin plays central roles in cellular function. The ribosomal DNA (rDNA) chromatin undergoes dynamic structural changes during mitosis and stress. Here, we developed a CRISPR-based imaging system and tracked the condensation dynamics of rDNA chromatin in live yeast cells under glucose starvation. We found that acute glucose starvation triggers rapid condensation of rDNA. Time-lapse microscopy revealed two stages for rDNA condensation: a "primary stage," when relaxed rDNA chromatin forms higher order loops or rings, and a "secondary stage," wehen the rDNA rings further condense into compact clusters. Twisting of rDNA rings accompanies the secondary stage. The condensin complex, but not the cohesin complex, is required for efficient rDNA condensation in response to glucose starvation. Furthermore, we found that the DNA helicase Sgs1 is essential for the survival of cells expressing rDNA-bound dCas9, suggesting a role for helicases in facilitating DNA replication at dCas9-binding sites.

Scarless genome editing: progress towards understanding genotype-phenotype relationships.

The ability to predict phenotype from genotype has been an elusive goal for the biological sciences for several decades. Progress decoding genotype-phenotype relationships has been hampered by the challenge of introducing precise genetic changes to specific genomic locations. Here we provide a comparative review of the major techniques that have been historically used to make genetic changes in cells as well as the development of the CRISPR technology which enabled the ability to make marker-free disruptions in endogenous genomic locations. We also discuss how the achievement of truly scarless genome editing has required further adjustments of the original CRISPR method. We conclude by examining recently developed genome editing methods which are not reliant on the induction of a DNA double strand break and discuss the future of both genome engineering and the study of genotype-phenotype relationships.