Sensing Inorganic Phosphate Starvation by the Phosphate-Responsive (PHO) Signaling Pathway of Saccharomyces cerevisiae

Inorganic phosphate $(P_i)$ is an essential nutrient whose intracellular levels are maintained by the PHO pathway in Saccharomyces cerevisiae. $P_i$ limitation triggers upregulation of the PHO genes whose gene products primarily function to counterbalance the $P_i$ deficiency. Despite a growing catalogue of genes that are involved in signaling of the PHO pathway, little is known about how cells actually sense $P_i$ limitation. To better characterize the $P_i$ sensing mechanism, I exploited two comprehensive and orthogonal approaches: 1) genome-wide genetic screening to identify novel genes involved in signaling $P_i$ limitation through the PHO pathway and characterization of genetic interactions among these genes and 2) liquid chromatography /mass spectrometry (LC/MS)-based metabolic profiling to characterize the metabolomic response to changes in $P_i$ availability. In genome-wide screening, I found that the aah1 mutant constitutively activated the PHO pathway and showed that AAH1 is involved in regulating PHO pathway activity. Moreover, I identified several novel genetic interactions of genes involved in inositol polyphosphate metabolism with those involved in purine metabolism and mitochondrial fatty acid biosynthesis.Through metabolomic profiling, I showed that all adenine nucleotides were downregulated in the constitutively induced ado1, adk1, and aah1 mutants in high $P_i$ as well as in the wild type strain in low $P_i$. These observations led to the hypothesis that downregulation of adenine nucleotides triggers activation of the PHO pathway. However, I find that decreases in adenine nucleotides appear to be the consequence of downregulation of glycolysis and of the pentose phosphate pathway rather than an activation signal for the PHO pathway.Among all the detected metabolites, S-adenosyl-L-homocysteine (SAH) responded the most quickly and significantly to changes in $P_i$ concentration. It was known that SAH is an inhibitor of de novo synthesis of phosphatidylcholine (PC). I showed that overall PC levels were downregulated in low $P_i$, suggesting that phospholipid metabolism is downregulated in low $P_i$ conditions. Furthermore, I observed that exogenous SAH induces activation of the PHO pathway in high $P_i$ implying a possible role of SAH as an initiating activation signal of the PHO pathway.Chemistry and Chemical Biolog

A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae

Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (P_i) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient P_i. P_i-limitation induces upregulation of inositol heptakisphosphate (IP_7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate the PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Furthermore, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of P_i starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated

On-chip Brillouin lasers based on 10 million-Q chalcogenide resonators without direct etch process

We present a new device platform which defines on-chip chalcogenide waveguide/resonators without directly etching chalcogenide. Using our platform, we have demonstrated chalcogenide ring resonators with record high Q-factor exceeding 1.1x107 which is 10 times larger than previous record on on-chip chalcogenide resonators. A ring cavity is designed and fabricated for Stimulated Brillouin lasing on our platform. Thanks to the high-Q factor, Brillouin lasing with threshold power of 1 mW is demonstrated. This value is more than an order of magnitude improvement than previous world record for on-chip chalcogenide Brillouin lasers. We also developed an efficient and flexible method for resonator waveguide coupling with our device platform. Coupling between a resonator and a waveguide can be varied from under coupled region to over-coupled regio

Universal light-guiding geometry for on-chip resonators having extremely high Q-factor

By providing an effective way to leverage nonlinear phenomena in integrated devices, high-Q optical resonators have led to recent advances in on-chip photonics. However, developing fabrication processes to shape any new material into a resonator with extremely smooth surfaces on a chip has been an exceptionally challenging task. Here, we describe a universal method to implement ultra-high-Q resonators with any new material having desirable properties that can be deposited by physical vapor deposition. Using this method light-guiding cores with surface roughness on the molecular-scale are created automatically on pre-patterned substrates. Its efficacy has been verified using As2S3, a chalcogenide glass that has high-nonlinearity. The Q-factor of the As2S3 resonator so-developed approached the propagation loss record achieved in chalcogenide fibers which were limited by material losses. Owing to the boosted Q-factor, lasing by stimulated Brillouin scattering has been demonstrated with 100 times lower threshold power than the previous record.This work was supported by Samsung Research Funding & Incubation Center of Samsung Electronics under Project Number SRFC-IT1801-03

Universal light-guiding geometry for on-chip resonators having extremely high Q-factor

By providing an effective way to leverage nonlinear phenomena in integrated devices, high-Q optical resonators have led to recent advances in on-chip photonics. However, developing fabrication processes to shape any new material into a resonator with extremely smooth surfaces on a chip has been an exceptionally challenging task. Here, we describe a universal method to implement ultra-high-Q resonators with any new material having desirable properties that can be deposited by physical vapor deposition. Using this method light-guiding cores with surface roughness on the molecular-scale are created automatically on pre-patterned substrates. Its efficacy has been verified using As2S3, a chalcogenide glass that has high-nonlinearity. The Q-factor of the As2S3 resonator so-developed approached the propagation loss record achieved in chalcogenide fibers which were limited by material losses. Owing to the boosted Q-factor, lasing by stimulated Brillouin scattering has been demonstrated with 100 times lower threshold power than the previous record. © 2020, The Author(s).1

Synthetic recording and in situ readout of lineage information in single cells

Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here, we describe a new synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out in single cells in situ. This system, termed Memory by Engineered Mutagenesis with Optical In situ Readout (MEMOIR), is based on a set of barcoded recording elements termed scratchpads. The state of a given scratchpad can be irreversibly altered by Cas9-based targeted mutagenesis, and read out in single cells through multiplexed single-molecule RNA fluorescence hybridization (smFISH). To demonstrate a proof of principle of MEMOIR, we engineered mouse embryonic stem (ES) cells to contain multiple scratchpads and other recording components. In these cells, scratchpads were altered in a progressive and stochastic fashion as cells proliferated. Analysis of the final states of scratchpads in single cells in situ enabled reconstruction of the lineage trees of cell colonies. Combining analysis of endogenous gene expression with lineage reconstruction in the same cells further allowed inference of the dynamic rates at which ES cells switch between two gene expression states. Finally, using simulations, we showed how parallel MEMOIR systems operating in the same cell can enable recording and readout of dynamic cellular event histories. MEMOIR thus provides a versatile platform for information recording and in situ, single cell readout across diverse biological systems

Synthetic recording and in situ readout of lineage information in single cells

Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here, we describe a new synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out in single cells in situ. This system, termed Memory by Engineered Mutagenesis with Optical In situ Readout (MEMOIR), is based on a set of barcoded recording elements termed scratchpads. The state of a given scratchpad can be irreversibly altered by Cas9-based targeted mutagenesis, and read out in single cells through multiplexed single-molecule RNA fluorescence hybridization (smFISH). To demonstrate a proof of principle of MEMOIR, we engineered mouse embryonic stem (ES) cells to contain multiple scratchpads and other recording components. In these cells, scratchpads were altered in a progressive and stochastic fashion as cells proliferated. Analysis of the final states of scratchpads in single cells in situ enabled reconstruction of the lineage trees of cell colonies. Combining analysis of endogenous gene expression with lineage reconstruction in the same cells further allowed inference of the dynamic rates at which ES cells switch between two gene expression states. Finally, using simulations, we showed how parallel MEMOIR systems operating in the same cell can enable recording and readout of dynamic cellular event histories. MEMOIR thus provides a versatile platform for information recording and in situ, single cell readout across diverse biological systems

Associations between the working experiences at frontline of COVID-19 pandemic and mental health of Korean public health doctors

Abstract Background Demographic, work environmental, and psychosocial features are associated with mental health of healthcare professionals at pandemic frontline. The current study aimed to find predictors of mental health for public health doctors from working experiences at frontline of COVID-19 pandemic. Methods With first-come and first-served manner, 350 public health doctors with experiences of work at COVID-19 frontline participated online survey on August 2020. Mental health was defined using the total scores of the Patient Health Questionnaire-9, the Generalized Anxiety Disorder-7, the Perceived Stress Scale, and the Stanford Presenteeism Scale-6. Multivariate logistic regression models of mental health with lowest Akaike Information Criterion were determined among all combinations of working environments, perceived threats and satisfaction at frontline, and demographics that were significant (P < 0.05) in the univariate logistic regression. Results Perceived distress, lowered self-efficacy at work, anxiety, and depressive mood were reported by 45.7, 34.6, 11.4, and 15.1% of respondents, respectively. Predictors of poor mental health found in the multivariate logistic regression analyses were environmental (insufficient personal protective equipment, workplace of screening center, prolonged workhours) and psychosocial (fear of infection and death, social stigma and rejection) aspects of working experiences at frontline. Satisfaction of monetary compensation and proactive coping (acceptance and willingness to volunteer at frontline) were predictive of better mental health. Conclusions Sufficient supply of personal protective equipment and training on infection prevention at frontline, proper workhours and satisfactory monetary compensation, and psychological supports are required for better mental health of public health doctors at frontline of COVID-19 pandemic