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

Scholarly publishing and argument in hyperspace

The World Wide Web is opening up access to documents and data for scholars. However it has not yet impacted on one of the primary activities in research: assessing new findings in the light of current knowledge and debating it with colleagues. The ClaiMaker system uses a directed graph model with similarities to hypertext, in which new ideas are published as nodes, which other contributors can build on or challenge in a variety of ways by linking to them. Nodes and links have semantic structure to facilitate the provision of specialist services for interrogating and visualizing the emerging network. By way of example, this paper is grounded in a ClaiMaker model to illustrate how new claims can be described in this structured way

Modelling naturalistic argumentation in research literatures: representation and interaction design issues

This paper characterises key weaknesses in the ability of current digital libraries to support scholarly inquiry, and as a way to address these, proposes computational services grounded in semiformal models of the naturalistic argumentation commonly found in research lteratures. It is argued that a design priority is to balance formal expressiveness with usability, making it critical to co-evolve the modelling scheme with appropriate user interfaces for argument construction and analysis. We specify the requirements for an argument modelling scheme for use by untrained researchers, describe the resulting ontology, contrasting it with other domain modelling and semantic web approaches, before discussing passive and intelligent user interfaces designed to support analysts in the construction, navigation and analysis of scholarly argument structures in a Web-based environment

From A to Z: Wearable technology explained

Wearable technology (WT) has become a viable means to provide low-cost clinically sensitive data for more informed patient assessment. The benefit of WT seems obvious: small, worn discreetly in any environment, personalised data and possible integration into communication networks, facilitating remote monitoring. Yet, WT remains poorly understood and technology innovation often exceeds pragmatic clinical demand and use. Here, we provide an overview of the common challenges facing WT if it is to transition from novel gadget to an efficient, valid and reliable clinical tool for modern medicine. For simplicity, an A–Z guide is presented, focusing on key terms, aiming to provide a grounded and broad understanding of current WT developments in healthcare

Psychosis risk candidate ZNF804A localizes to synapses and regulates neurite formation and dendritic spine structure

BackgroundVariation in the gene encoding zinc finger binding protein 804A (ZNF804A) is associated with schizophrenia (SCZ) and bipolar disorder (BP). Evidence suggests that ZNF804A is a regulator of gene transcription and is present in nuclear and extranuclear compartments. However, a detailed examination of ZNF804A distribution and its neuronal functions has yet to be performed.MethodsThe localization of ZNF804A protein was examined in neurons derived from human neural progenitor cells (hNPCs), human induced pluripotent stem cells (hiPSCs) or in primary rat cortical neurons. Additionally, siRNA-mediated knockdown of ZNF804A was conducted to determine its role in neurite formation, maintenance of dendritic spine morphology and responses to activity-dependent stimulations.ResultsEndogenous ZNF804A protein localized to somato-dendritic compartments and co-localized with the putative synaptic markers in young neurons derived from hNPCs and hiPSCs. In mature rat neurons, Zfp804A, the homolog of ZNF804A, was present in a subset of dendritic spines and co-localized with synaptic proteins in specific nanodomains, as determined by superresolution microscopy. Interestingly, knockdown of ZNF804A attenuated neurite outgrowth in young neurons, an effect potentially mediated by reduced neuroligin-4 (NLGN4) expression. Furthermore, knockdown of ZNF804A in mature neurons resulted in the loss of dendritic spine density, and impaired responses to activity-dependent stimulation.ConclusionsThese data reveal a novel subcellular distribution for ZNF804A within somato-dendritic compartments and a nanoscopic organisation at excitatory synapses. Moreover, our results suggest that ZNF804A plays an active role in neurite formation, maintenance of dendritic spines and activity-dependent structural plasticity

Mouse hitchhiker mutants have spina bifida, dorso-ventral patterning defects and polydactyly: identification of Tulp3 as a novel negative regulator of the Sonic hedgehog pathway

The mammalian Sonic hedgehog (Shh) signalling pathway is essential for embryonic development and the patterning of multiple organs. Disruption or activation of Shh signalling leads to multiple birth defects, including holoprosencephaly, neural tube defects and polydactyly, and in adults results in tumours of the skin or central nervous system. Genetic approaches with model organisms continue to identify novel components of the pathway, including key molecules that function as positive or negative regulators of Shh signalling. Data presented here define Tulp3 as a novel negative regulator of the Shh pathway. We have identified a new mouse mutant that is a strongly hypomorphic allele of Tulp3 and which exhibits expansion of ventral markers in the caudal spinal cord, as well as neural tube defects and preaxial polydactyly, consistent with increased Shh signalling. We demonstrate that Tulp3 acts genetically downstream of Shh and Smoothened (Smo) in neural tube patterning and exhibits a genetic interaction with Gli3 in limb development. We show that Tulp3 does not appear to alter expression or processing of Gli3, and we demonstrate that transcriptional regulation of other negative regulators (Rab23, Fkbp8, Thm1, Sufu and PKA) is not affected. We discuss the possible mechanism of action of Tulp3 in Shh-mediated signalling in light of these new data

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Antibodies against endogenous retroviruses promote lung cancer immunotherapy

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS). Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

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