Phosphatidylinositol (4,5)-bisphosphate turnover by INP51 regulates the cell wall integrity pathway in "Saccharomyces cerevisiae"

Signal transduction pathways are important for the cell to transduce external or internal stimuli where second messengers play an important role as mediators of the stimuli. One important group of second messengers are the phosphoinositide family present in organisms ranging from yeast to mammals. The dephosphorylation and phosphorylation cycle of the phosphatidylinositol species are thought to be important in signaling for recruitment or activation of proteins involved in vesicular transport and/or to control the organization of the actin cytoskeleton. In mammals, phosphatidylinositol (4,5)bisphosphate (PI(4,5)P2) signaling is essential and regulated by various kinases and phosphatases. In the model organism Saccharomyces cerevisiae PI(4,5)P2 signaling is also essential but the regulation remains unclear. My dissertation focuses on the regulation of PI(4,5)P2 signaling in Saccharomyces cerevisiae. The organization of the actin cytoskeleton in Saccharomyces cerevisiae is regulated by different proteins such as calmodulin, CMD1, and here I present data that CMD1 plays a role in the regulation of the only phosphatidylinositol 4-phosphate 5-kinase, MSS4, in Saccharomyces cerevisiae. CMD1 regulates MSS4 activity through an unknown mechanism and thereby controls the organization of the actin cytoskeleton. MSS4 and CMD1 do not physically interact but MSS4 seems to be part of a large molecular weight complex as shown by gel filtration chromatography. This complex could contain regulators of the MSS4 activity. The complex is not caused by dimerization of MSS4 since MSS4 does not interact with itself. Two pathways, the cell wall integrity pathway and TORC2 (target of rapamycin complex 2) signaling cascade are important for the organization of the actin cytoskeleton. Loss of TOR2 function results in a growth defect that can be suppressed by MSS4 overexpression. To further characterize the link between MSS4 and the TORC2 signaling pathway and the cell wall integrity pathway we looked for targets of PI(4,5)P2. The TORC2 pathway and the cell wall integrity pathway signal to the GEF ROM2, an activator of the small GTPase RHO1. In our study we identified ROM2 as a target of PI(4,5)P2 signaling. We observed that the ROM2 localization changes in an mss4 conditional mutant. This suggests that the proper localization needs PI(4,5)P2. This could be mediated by the putative PI(4,5)P2 binding pleckstrin homology (PH) domain of ROM2. To better understand the regulation of PI(4,5)P2 levels in Saccharomyces cerevisiae we focused on one of the PI(4,5)P2 5-phosphatases, INP51. Here we present evidence that INP51 is a new negative regulator of the cell wall integrity pathway as well as the TORC2 pathway. INP51 probably regulates these two pathways by the turnover of PI(4,5)P2 thereby inactivating the effector/s. The deletion of INP51 does not result in any phenotype, but when combined with mutations of the cell wall integrity pathway we observe synthetic interaction. INP51 together with the GTPase activating protein (GAP) SAC7, responsible for the negative regulation of RHO1, negatively regulates the cell wall integrity pathway during vegetative growth. One of the targets of cell wall integrity pathway, the cell wall component chitin, which is normally deposited at the bud end, bud neck and forms bud scars, is delocalized in the mother cell in the sac7 inp51 double deletion mutant. In addition, another downstream component of the cell wall integrity pathway, the MAP kinase MPK1, has increased phosphorylation and protein level in the sac7 inp51 double deletion mutant. This suggests that INP51 is important for the negative regulation of the cell wall integrity pathway. Furthermore, we show evidence that INP51 forms a complex with TAX4 or IRS4, with two EH-domain containing proteins, that positively regulates the activity of INP51 and in this manner negatively regulate the cell wall integrity pathway. The EH-domain is known to bind the NPF-motif. This motif is present in INP51 and is important for INP51 interaction with TAX4 or IRS4. The EH-NPF interaction is a conserved mechanism to build up protein networks. The interaction between an EH-domain containing protein and a PI(4,5)P2 5-phosphatase is conserved. This is demonstrated by the epidermal growth factor substrate EPS15 (EH) interaction with the PI(4,5)P2 5-phosphatase synaptojanin the mammalian orthologue of the Saccharomyces cerevisiae INP proteins. In summary, INP51 together with TAX4 and IRS4, forms complexes important for regulation of PI(4,5)P2 levels. The complexes are linked to the TORC2 signaling pathway and the cell wall integrity pathway, specifically regulating MPK1 activation and chitin biosynthesis. The work presented in this dissertation facilitates the development of a model of the complex regulation of PI(4,5)P2 signaling in Saccharomyces cerevisiae

Human Protein Phosphatase PP6 Regulatory Subunits Provide Sit4-Dependent and Rapamycin–Sensitive Sap Function in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae the protein phosphatase Sit4 and four associated proteins (Sap4, Sap155, Sap185, and Sap190) mediate G1 to S cell cycle progression and a number of signaling events controlled by the target of rapamycin TOR signaling cascade. Sit4 and the Sap proteins are ubiquitously conserved and their human orthologs, PP6 and three PP6R proteins, share significant sequence identity with their yeast counterparts. However, relatively little is known about the functions of the PP6 and PP6R proteins in mammalian cells. Here we demonstrate that the human PP6R proteins physically interact with Sit4 when expressed in yeast cells. Remarkably, expression of PP6R2 and PP6R3 but not expression of PP6R1 rescues the growth defect and rapamycin hypersensitivity of yeast cells lacking all four Saps, and these effects require Sit4. Moreover, PP6R2 and PP6R3 enhance cyclin G1 gene expression and DNA synthesis, and partially abrogate the G1 cell cycle delay and the budding defect of the yeast quadruple sap mutant strain. In contrast, the human PP6R proteins only modestly support nitrogen catabolite gene expression and are unable to restore normal levels of eIF2α phosphorylation in the quadruple sap mutant strain. These results illustrate that the human PP6-associated proteins are capable of providing distinct rapamycin-sensitive and Sit4-dependent Sap functions in the heterologous context of the yeast cell. We hypothesize that the human Saps may play analogous roles in mTORC1-PP6 signaling events in metazoans

Colloidal Flower-Shaped Iron Oxide Nanoparticles: Synthesis Strategies and Coatings

The assembly of magnetic cores into regular structures may notably influence the properties displayed by a magnetic colloid. Here, key synthesis parameters driving the self-assembly process capable of organizing colloidal magnetic cores into highly regular and reproducible multi-core nanoparticles are determined. In addition, a self-consistent picture that explains the collective magnetic properties exhibited by these complex assemblies is achieved through structural, colloidal, and magnetic means. For this purpose, different strategies to obtain flower-shaped iron oxide assemblies in the size range 25-100 nm are examined. The routes are based on the partial oxidation of Fe(OH)(2), polyol-mediated synthesis or the reduction of iron acetylacetonate. The nanoparticles are functionalized either with dextran, citric acid, or alternatively embedded in polystyrene and their long-term stability is assessed. The core size is measured, calculated, and modeled using both structural and magnetic means, while the Debye model and multi-core extended model are used to study interparticle interactions. This is the first step toward standardized protocols of synthesis and characterization of flower-shaped nanoparticles

2D versus 3D human induced pluripotent stem cell-derived cultures for neurodegenerative disease modelling

Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS), affect millions of people every year and so far, there are no therapeutic cures available. Even though animal and histological models have been of great aid in understanding disease mechanisms and identifying possible therapeutic strategies, in order to find disease-modifying solutions there is still a critical need for systems that can provide more predictive and physiologically relevant results. One possible avenue is the development of patient-derived models, e.g. by reprogramming patient somatic cells into human induced pluripotent stem cells (hiPSCs), which can then be differentiated into any cell type for modelling. These systems contain key genetic information from the donors, and therefore have enormous potential as tools in the investigation of pathological mechanisms underlying disease phenotype, and progression, as well as in drug testing platforms. hiPSCs have been widely cultured in 2D systems, but in order to mimic human brain complexity, 3D models have been proposed as a more advanced alternative. This review will focus on the use of patient-derived hiPSCs to model AD, PD, HD and ALS. In brief, we will cover the available stem cells, types of 2D and 3D culture systems, existing models for neurodegenerative diseases, obstacles to model these diseases in vitro, and current perspectives in the field

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

Global age-sex-specific fertility, mortality, healthy life expectancy (HALE), and population estimates in 204 countries and territories, 1950-2019 : a comprehensive demographic analysis for the Global Burden of Disease Study 2019

Background: Accurate and up-to-date assessment of demographic metrics is crucial for understanding a wide range of social, economic, and public health issues that affect populations worldwide. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 produced updated and comprehensive demographic assessments of the key indicators of fertility, mortality, migration, and population for 204 countries and territories and selected subnational locations from 1950 to 2019. Methods: 8078 country-years of vital registration and sample registration data, 938 surveys, 349 censuses, and 238 other sources were identified and used to estimate age-specific fertility. Spatiotemporal Gaussian process regression (ST-GPR) was used to generate age-specific fertility rates for 5-year age groups between ages 15 and 49 years. With extensions to age groups 10–14 and 50–54 years, the total fertility rate (TFR) was then aggregated using the estimated age-specific fertility between ages 10 and 54 years. 7417 sources were used for under-5 mortality estimation and 7355 for adult mortality. ST-GPR was used to synthesise data sources after correction for known biases. Adult mortality was measured as the probability of death between ages 15 and 60 years based on vital registration, sample registration, and sibling histories, and was also estimated using ST-GPR. HIV-free life tables were then estimated using estimates of under-5 and adult mortality rates using a relational model life table system created for GBD, which closely tracks observed age-specific mortality rates from complete vital registration when available. Independent estimates of HIV-specific mortality generated by an epidemiological analysis of HIV prevalence surveys and antenatal clinic serosurveillance and other sources were incorporated into the estimates in countries with large epidemics. Annual and single-year age estimates of net migration and population for each country and territory were generated using a Bayesian hierarchical cohort component model that analysed estimated age-specific fertility and mortality rates along with 1250 censuses and 747 population registry years. We classified location-years into seven categories on the basis of the natural rate of increase in population (calculated by subtracting the crude death rate from the crude birth rate) and the net migration rate. We computed healthy life expectancy (HALE) using years lived with disability (YLDs) per capita, life tables, and standard demographic methods. Uncertainty was propagated throughout the demographic estimation process, including fertility, mortality, and population, with 1000 draw-level estimates produced for each metric. Findings: The global TFR decreased from 2·72 (95% uncertainty interval [UI] 2·66–2·79) in 2000 to 2·31 (2·17–2·46) in 2019. Global annual livebirths increased from 134·5 million (131·5–137·8) in 2000 to a peak of 139·6 million (133·0–146·9) in 2016. Global livebirths then declined to 135·3 million (127·2–144·1) in 2019. Of the 204 countries and territories included in this study, in 2019, 102 had a TFR lower than 2·1, which is considered a good approximation of replacement-level fertility. All countries in sub-Saharan Africa had TFRs above replacement level in 2019 and accounted for 27·1% (95% UI 26·4–27·8) of global livebirths. Global life expectancy at birth increased from 67·2 years (95% UI 66·8–67·6) in 2000 to 73·5 years (72·8–74·3) in 2019. The total number of deaths increased from 50·7 million (49·5–51·9) in 2000 to 56·5 million (53·7–59·2) in 2019. Under-5 deaths declined from 9·6 million (9·1–10·3) in 2000 to 5·0 million (4·3–6·0) in 2019. Global population increased by 25·7%, from 6·2 billion (6·0–6·3) in 2000 to 7·7 billion (7·5–8·0) in 2019. In 2019, 34 countries had negative natural rates of increase; in 17 of these, the population declined because immigration was not sufficient to counteract the negative rate of decline. Globally, HALE increased from 58·6 years (56·1–60·8) in 2000 to 63·5 years (60·8–66·1) in 2019. HALE increased in 202 of 204 countries and territories between 2000 and 2019

<i>In vitro</i> culture of adult human alveolar epithelial cells.

<p>(A) Human lung AT2 cell morphology changes more when cultured on rat-tail collagen-coated dishes than on Matrigel-coated dishes. Freshly-isolated human AT2 cells were seeded on collagen-coated or Matrigel-coated tissue culture dishes and photomicrographs were acquired after 12 hours (“Day 0”), 1, 2, and 3 days. Cells on collagen flattened and spread; cells on Matrigel remained cuboidal in shape and accumulated into enlarging “cysts”. Original magnification, 100X. (B) Gene expression analysis of Day 3 cells shows that SP-C, a marker of AT2 cells, is not expressed in cells cultured on collagen; however, its expression is retained on Matrigel.</p

Whole-Genome Analysis of Temporal Gene Expression during Early Transdifferentiation of Human Lung Alveolar Epithelial Type 2 Cells <i>In Vitro</i>

<div><p>It is generally accepted that the surfactant-producing pulmonary alveolar epithelial type II (AT2) cell acts as the progenitor of the type I (AT1) cell, but the regulatory mechanisms involved in this relationship remain the subject of active investigation. While previous studies have established a number of specific markers that are expressed during transdifferentiation from AT2 to AT1 cells, we hypothesized that additional, previously unrecognized, signaling pathways and relevant cellular functions are transcriptionally regulated at early stages of AT2 transition. In this study, a discovery-based gene expression profile analysis was undertaken of freshly isolated human AT2 (hAT2) cells grown on extracellular matrix (ECM) substrata known to either support (type I collagen) or retard (Matrigel) the early transdifferentiation process into hAT1-like cells over the first three days. Cell type-specific expression patterns analyzed by Illumina Human HT-12 BeadChip yielded over 300 genes that were up- or down-regulated. Candidate genes significantly induced or down-regulated during hAT2 transition to hAT1-like cells compared to non-transitioning hAT2 cells were identified. Major functional groups were also recognized, including those of signaling and cytoskeletal proteins as well as genes of unknown function. Expression of established signatures of hAT2 and hAT1 cells, such as surfactant proteins, caveolin-1, and channels and transporters, was confirmed. Selected novel genes further validated by qRT-PCR, protein expression analysis, and/or cellular localization included SPOCK2, PLEKHO1, SPRED1, RAB11FIP1, PTRF/CAVIN-1 and RAP1GAP. These results further demonstrate the utility of genome-wide analysis to identify relevant, novel cell type-specific signatures of early ECM-regulated alveolar epithelial transdifferentiation processes in vitro.</p></div

Time course protein expression analysis of selected candidate genes.

<p>SPOCK2 and PTRF/CAVIN-1 proteins increase in transitioning hAT2 cells on collagen over 10 days, while Rap1GAP is expressed mainly in AT2 cells on Matrigel. AT1 signature caveolin-1 and AT2 signature SP-D are shown to confirm phenotype. Freshly-isolated hAT2 cells were seeded on rat-tail collagen or Matrigel; cells were harvested for proteins on days 1, 2, 3, 6 and 10 after seeding. Results shown are representative of three separate time course experiments using cells from two isolations.</p

Validation of a subset of candidate genes by qRT-PCR.

<p>RNA was harvested from hAT2 cells cultured on collagen or Matrigel at 12 = 2. The relative expressions of SPOCK2, PLEKHO1, SPRED1, RAB11FIP1, PTRF/CAVIN-1, and RAP1GAP were assessed by TaqMan qRT-PCR and normalized to GAPDH and TBP. The fold-changes in relative expression over time are shown as graphs. The smaller, inset graphs show the expression results obtained from the Illumina BeadChip analysis, for comparison.</p