Analysis of Histone Deacetylase Involvement in \u3ci\u3ePseudomonas syringae\u3c/i\u3e-triggered Chromatin Changes

I had the great privilege of working with Dr. Karin van Dijk, through UCARE, researching the specific pathways through which histone deacetylation occurs in plants during the 2015-2016 academic year. My first year conducting research helped me develop my technical skills and learn about experimental protocols and how to conduct them. I would like to continue my research during the 2016-2017 academic year and now that I have a year of research experience, be able to further develop my skills and learn more about my research project. Background Pseudomonas syringae is a bacterial pathogen that is well known for causing bacterial speck disease in various hosts, including the economically relevant crops, tomato and soybean. One of the primary mechanisms used by P. syringae to cause disease is the injection of a plethora of effector proteins via the type 3 secretion system (T3SS) into the plant host cells. Although we know these proteins collectively enable the pathogen to cause disease by primarily disabling or subverting immune defenses, the specific details of how each effector protein does this is not very well understood for the majority of effector proteins. However, we do know that very quickly after infection, changes in host gene expression can be detected. Studies in the laboratory of Dr. Karin van Dijk found that there is a rapid deacetylation of host histone H3 lysine 9 (H3K9) upon infection with P. syringae. The deacetylation of H3k9ac was found to be located along a number of innate immune genes, and a correlative reduction of gene expression was observed as well. This reduced acetylation was found to depend on a functional T3SS and the effectors traveling through it. We believe this effector-driven deacetylation of H3K9 is involved in the impairment of the plants immune response to the pathogen, which enables the pathogen to cause disease. The purpose of this research is to investigate the molecular mechanism by which the effector-dependent deacetylation occurs. It is possible the type III effectors (T3E) trigger an expression change in one or more known histone acetyltransferases (HATs) and/or histone deacetylases (HDACs). To begin to explore this, we have performed qRT-PCR on plant samples exposed to the pathogen or not to measure expression levels of several known HATs and HDACs. We found several HDACs that are upregulated in plants exposed to the pathogen and will focus our research on these. This research is important because it will allow us to help identify the mechanism by which P. syringae suppresses immunity and thus cause disease in staple crops like soybeans. This may help aid in the development of products to protect these plants from infection. Purpose We hypothesize that the effector-driven deacetylation occurs through upregulation of a plant-encoded Histone deacetylase that deacetylases H3K9ac. I will be working on this research in cooperation with another undergraduate student, who is approaching this research from the perspective of bacterial effector proteins. I will not work directly with her, but our two research questions overlap and the findings from each study will help in understanding our own projects. Procedures For this research I will use T-DNA lines of Arabidopsis defective in specific HDACs. I will analyze these plants for their ability/inability to deacetylate H3K9ac along innate immune genes once exposed to the pathogen. I will also use these plants in pathogenicity assays to determine their susceptibility to P. syringae. I will begin this experiment by growing wildtype and mutant Arabidopsis plants under similar conditions to allow for as little growth deviation as possible. Once the plants are fully grown, I will separate the plants into different groups. One group will be the control group and will get infiltrated with a buffer solution. Another group will be the group of plants exposed to wild type P. syringae. The last group will be exposed to a mutant strain of P. syringae unable to produce the T3SS. If used in pathogenicity assays, growth of the bacterial strains will be followed over a 6-day period by plating leaf samples on media selective for P. syringae. To analyze chromatin deacetylation, we will use two different techniques, immunoblotting and Chromatin Immunoprecipitation combined with quantitative PCR (ChIP-qPCR). After exposure, the leaves from each of the plants in each group will be harvested. For the samples that will be analyzed by immunoblotting, we will flash freeze these leaves in liquid nitrogen and store the tissue at -80 until use. For the ChIP-qPCR analysis, Chromatin will be cross-linked with formaldehyde and the leaves flash frozen and stored at -80 until use. For the data analysis, we will use immunoblot analysis of leaf tissue to determine if the mutant plants can still deacetylate H3K9ac upon P. syringae infection. We will separate proteins isolated from leaf tissue on SDS-PAGE gels, blot the proteins onto membranes and use anti-H3K9ac antibodies and anti-H3 antibodies to detect H3 acetylateion at K9 and total H3 acetylation, respectively. We will use densitometry to determine relative acetylation of H3K9. This will allow us to compare acetylation levels between the different Arabidopsis lines. I will use ChIP-qPCR in order to determine if deacetylation happened along innate immune genes. To do so, chromatin will be prepared from frozen cross-linked plant tissues. Next the chromatin will be sheared into small fragments (averaging 500 bp) and precipitated using either anti-H3K9ac or anti-H3 antibodies. Precipitated chromatin will be decross-linked and the released DNA will be analyzed with qPCR using primers to amplify specific innate immune genes. By comparing relative PCR product amounts between strains, I will be able to determine if there is a change in the H3K9 acetylation along these innate immune genes in the mutant Arabidopsis lines relative to the wildtype. Benchmarks: Plant Arabidopsis plants Conduct library research/literature review Conduct research (gather data) Inject plants with P. syringae Collect leaves Prepare pathogenicity assays Perform immunoblot analysis on the three groups of leaves Use ChIP-qPCR on the three leaf groups Analyze data Determine acetylation of H3K9ac after infection of P. syringae in all three plant groups using densitrometry Determine if deacetylation occurred from ChIP-qPCR Present findings at Nebraska Academy of Science Present findings at UCARE presentation Some of the benchmarks mentioned above have been achieved during the 2015-2016 academic year through UCARE, such as conducting research by injecting plants with P. syringae, collecting leaves, preparing pathogenicity assays, performing immunoblot analysis on the three groups of leaves, and also determining the acetylation of H3K9ac after infection of P. syringae in all three plant groups using densitrometry. During the academic year I hope to continue with my research by performing ChIP-qPCR on our leaf samples and analyzing the data to determine if deacetylation has occurred

Real bad grammar: realistic grammatical description with grammaticality

Sampson (this issue) argues for a concept of “realistic grammatical description” in which the distinction between grammatical and ungrammatical sentences is irrelevant. In this article I also argue for a concept of “realistic grammatical description” but one in which a binary distinction between grammatical and ungrammatical sentences is maintained. In distinguishing between the grammatical and ungrammatical, this kind of grammar differs from that proposed by Sampson, but it does share the important property that invented sentences have no role to play, either as positive or negative evidence

An economic model for offshore cultivation of macroalgae

Algae biomass is considered as a potential non-fossil source of raw materials to produce fuel, feed, chemicals and materials. For this purpose microalgae as well as macroalgae can be used, and in this report we focus on the latter. More than 99% of the world production of aquatic plants is produced in Asia (FAO 2012, Table 1). From the remaining 1% about 4% is cultivated in Europe. Important European countries with commercial seaweed cultivation are Denmark, Ireland and France. Depending on their pigmentation seaweed species are commonly grouped in brown, red and green seaweeds

Analysis of the Brinkman-Forchheimer equations with slip boundary conditions

In this work, we study the Brinkman-Forchheimer equations driven under slip boundary conditions of friction type. We prove the existence and uniqueness of weak solutions by means of regularization combined with the Faedo-Galerkin approach. Next we discuss the continuity of the solution with respect to Brinkman's and Forchheimer's coefficients. Finally, we show that the weak solution of the corresponding stationary problem is stable

Psychosocial function of Dutch children with cancer and their caregivers during different phases of the COVID-19 pandemic

We compared psychosocial functioning of children with cancer and their caregivers in several phases of the coronavirus disease 2019 (COVID-19) pandemic to before COVID-19. One or more questionnaires on health-related quality of life (HRQoL) or fatigue of children or distress of their caregivers was available from 1644 families. In children with cancer, HRQoL was stable throughout the COVID-19 pandemic. Fatigue was slightly lower and sleep somewhat better during the pandemic than before. Caregiver distress was lower in the first pandemic phase, but increased to pre-COVID-19 levels in later phases, indicating that the length and consequences of the pandemic may be weighing on them

The movement ecology of seagrasses

A movement ecology framework is applied to enhance our understanding of the causes, mechanisms and consequences of movement in seagrasses: marine, clonal, flowering plants. Four life-history stages of seagrasses can move: pollen, sexual propagules, vegetative fragments and the spread of individuals through clonal growth. Movement occurs on the water surface, in the water column, on or in the sediment, via animal vectors and through spreading clones. A capacity for long-distance dispersal and demographic connectivity over multiple timeframes is the novel feature of the movement ecology of seagrasses with significant evolutionary and ecological consequences. The space–time movement footprint of different life-history stages varies. For example, the distance moved by reproductive propagules and vegetative expansion via clonal growth is similar, but the timescales range exponentially, from hours to months or centuries to millennia, respectively. Consequently, environmental factors and key traits that interact to influence movement also operate on vastly different spatial and temporal scales. Six key future research areas have been identified

The dynamics of ovine gastrointestinal nematode infections within ewe and lamb cohorts on three Scottish sheep farms

Gastrointestinal nematodes (GIN) are a serious concern for sheep producers worldwide. However, there is a paucity of evidence describing the epidemiology of GIN on modern UK sheep farms. The aim of this paper was to understand whether expected seasonal variations of infection are still found in ewes and lambs under varying management strategies in temperate climates. Faecal egg counts (FEC) were conducted on freshly voided samples collected from groups of ewes and lambs every third week for twelve months on three farms in southeast Scotland. The patterns of egg output have been described here in relation to management practices undertaken on the farms. Despite changes in farming practice and climatic conditions, the findings complement historical studies detailing the epidemiology of GIN. Findings include a periparturient rise in ewe FEC on two of the farms, while lambing time treatment appeared to suppress this on the third farm. On the same two farms lamb FEC increased during the summer, reaching a peak in the autumn. The work also highlights how the ad hoc use of anthelmintics does little to impact these patterns

Nonadherence in hemodialysis: Associations with mortality, hospitalization, and practice patterns in the DOPPS

Nonadherence in hemodialysis: Associations with mortality, hospitalization, and practice patterns in the DOPPS.BackgroundNonadherence among hemodialysis patients compromises dialysis delivery, which could influence patient morbidity and mortality. The Dialysis Outcomes and Practice Patterns Study (DOPPS) provides a unique opportunity to review this problem and its determinants on a global level.MethodsNonadherence was studied using data from the DOPPS, an international, observational, prospective hemodialysis study. Patients were considered nonadherent if they skipped one or more sessions per month, shortened one or more sessions by more than 10 minutes per month, had a serum potassium level openface>6.0mEq/L, a serum phosphate level openface>7.5mg/dL (>2.4mmol/L), or interdialytic weight gain (IDWG)>5.7% of body weight. Predictors of nonadherence were identified using logistic regression. Survival analysis used the Cox proportional hazards model adjusting for case-mix.ResultsSkipping treatment was associated with increased mortality [relative risk (RR) = 1.30, P = 0.01], as were excessive IDWG (RR = 1.12, P = 0.047) and high phosphate levels (RR = 1.17, P = 0.001). Skipping also was associated with increased hospitalization (RR = 1.13, P = 0.04), as were high phosphate levels (RR = 1.07, P = 0.05). Larger facility size (per 10 patients) was associated with higher odds ratios (OR) of skipping (OR = 1.03, P = 0.06), shortening (OR = 1.03, P = 0.05), and IDWG (OR = 1.02, P = 0.07). An increased percentage of highly trained staff hours was associated with lower OR of skipping (OR = 0.84 per 10%, P = 0.02); presence of a dietitian was associated with lower OR of excessive IDWG (OR = 0.75, P = 0.08).ConclusionNonadherence was associated with increased mortality risk (skipping treatment, excessive IDWG, and high phosphate) and with hospitalization risk (skipping, high phosphate). Certain patient/facility characteristics also were associated with nonadherence

A novel missense variant in ATP11CATP11C is associated with reduced red blood cell phosphatidylserine flippase activity and mild hereditary hemolytic anemia

Adenosine Triphosphatase (ATPase) Phospholipid Transporting 11C gene (ATP11C) encodes the major phosphatidylserine (PS) flippase in human red blood cells (RBCs). Flippases actively transport phospholipids (e.g., PS) from the outer to the inner leaflet to establish and maintain phospholipid asymmetry of the lipid bilayer of cell membranes. This asymmetry is crucial for survival since externalized PS triggers phagocytosis by splenic macrophages. Here we report on pathophysiological consequences of decreased flippase activity, prompted by a patient with hemolytic anemia and hemizygosity for a novel c.2365C > T p.(Leu789Phe) missense variant in ATP11C. ATP11C protein expression was strongly reduced by 58% in patient‐derived RBC ghosts. Furthermore, functional characterization showed only 26% PS flippase activity. These results were confirmed by recombinant mutant ATP11C protein expression in HEK293T cells, which was decreased to 27% compared to wild type, whereas PS‐stimulated ATPase activity was decreased by 57%. Patient RBCs showed a mild increase in PS surface exposure when compared to control RBCs, which further increased in the most dense RBCs after RBC storage stress. The increase in PS was not due to higher global membrane content of PS or other phospholipids. In contrast, membrane lipid lateral distribution showed increased abundance of cholesterol‐enriched domains in RBC low curvature areas. Finally, more dense RBCs and subtle changes in RBC morphology under flow hint toward alterations in flow behavior of ATP11C‐deficient RBCs. Altogether, ATP11C deficiency is the likely cause of hemolytic anemia in our patient, thereby underlining the physiological role and relevance of this flippase in human RBCs