Dueling Electrospray Implemented on a Traveling-Wave Ion Mobility/Time-Of-Flight Mass Spectrometer: Towards a Gas-Phase Workbench for Structural Biology

• Covalent chemistry was performed in an ion mobility/mass spectrometer (IM/MS). • The effects of various parameters on the extent of reactions was observed. • Angiotensin I and ubiquitin were covalently labeled in the gas-phase. • Ion/ion reactions modify ions with solution-like conformations.The traveling wave trap cell of a commercial ion mobility mass spectrometer (IM/MS) was used as a gas-phase reactor for covalent chemistry by making a simple modification to a standard nanoelectrospray source. Reagents and analytes were generated from pulsed opposite polarity nanoelectrospray sources and isolated by their m/z prior to reaction. Covalent bond formation was first observed with the model peptide angiotensin I. The modification site was identified as the N-terminus of the peptide by collision induced dissociation (CID). The IM cell separated the covalent reaction product from the proton transfer product by their respective ion mobilities. Next, the effects of several trapping parameters, including the trap traveling wave height, the trap RF voltage, and the trap pressure, were evaluated. Decreasing traveling wave height and increasing RF voltage and pressure increased the number of proton transfer events from apomyoglobin to reagent anions. The 6+ charge state of ubiquitin generated from nanospray under native-like conditions was covalently modified in the gas phase through ion/ion reactions. Probing the reacted protein with CID led to the assignment of lysine 29 and arginine 54 as reactive nucleophiles accessible to the reagent. IM analysis of the unmodified native-like 6+ charge state revealed that the gas-phase structure of the protein in the trap was in its compact form. Overall, we introduce a promising method for three-dimensional structural characterization of biomacromolecules

PRMT5-Selective Inhibitors Suppress Inflammatory T Cell Responses and Experimental Autoimmune Encephalomyelitis

Poster Division: Biological Sciences: 2nd Place (The Ohio State University Edward F. Hayes Graduate Research Forum)In the autoimmune disease multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), ex- pansion of pathogenic, myelin-specific Th1 cell populations drives active disease; selectively targeting this process may be the basis for a new therapeutic approach. Previous studies have hinted at a role for protein arginine methylation in immune responses, in- cluding T cell–mediated autoimmunity and EAE. However, a conclusive role for the protein arginine methyltransferase (PRMT) enzymes that catalyze these reactions has been lacking. PRMT5 is the main PRMT responsible for symmetric dimethylation of arginine residues of histones and other proteins. PRMT5 drives embryonic development and cancer, but its role in T cells, if any, has not been investigated. In this article, we show that PRMT5 is an important modulator of CD4+ T cell expansion. PRMT5 was transiently upregulated during maximal proliferation of mouse and human memory Th cells. PRMT5 expression was regulated upstream by the NF-kB pathway, and it promoted IL-2 production and proliferation. Blocking PRMT5 with novel, highly selective small molecule PRMT5 inhibitors severely blunted memory Th expansion, with preferential suppression of Th1 cells over Th2 cells. In vivo, PRMT5 blockade efficiently suppressed recall T cell responses and reduced inflammation in delayed-type hypersensitivity and clinical disease in EAE mouse models. These data implicate PRMT5 in the regulation of adaptive memory Th cell responses and suggest that PRMT5 inhibitors may be a novel therapeutic approach for T cell–mediated inflammatory disease. The Journal of Immunology, 2017, 198: 000–000.A three-year embargo was granted for this item

Effects of regional differences and demography in modelling foot-and-mouth disease in cattle at the national scale

Foot-and-mouth disease (FMD) is a fast-spreading viral infection that can produce large and costly outbreaks in livestock populations. Transmission occurs at multiple spatial scales, as can the actions used to control outbreaks. The US cattle industry is spatially expansive, with heterogeneous distributions of animals and infrastructure. We have developed a model that incorporates the effects of scale for both disease transmission and control actions, applied here in simulating FMD outbreaks in US cattle. We simulated infection initiating in each of the 3049 counties in the contiguous US, 100 times per county. When initial infection was located in specific regions, large outbreaks were more likely to occur, driven by infrastructure and other demographic attributes such as premises clustering and number of cattle on premises. Sensitivity analyses suggest these attributes had more impact on outbreak metrics than the ranges of estimated disease parameter values. Additionally, although shipping accounted for a small percentage of overall transmission, areas receiving the most animal shipments tended to have other attributes that increase the probability of large outbreaks. The importance of including spatial and demographic heterogeneity in modelling outbreak trajectories and control actions is illustrated by specific regions consistently producing larger outbreaks than others

ENGAGEMENT MATTERS: An exploration of public engagement and its futures in Toronto

As public engagement gains momentum as a mechanism for engaging residents in a changing political climate, its effectiveness is more important than ever. This study, divided into three volumes, explores matters related to public engagement for city-building decisions. Volume 1 is a primer on public engagement, meant to support municipal public servants to understand the basics of public engagement. This Volume is informed by user and expert interviews, ethnographic observation, and system mapping tools conducted during the first phase of this research. Volume 2 builds on insights developed during the first phase of this research, to explore the futures of public engagement in 2033 using strategic foresight. Written for public servants familiar with the field of public engagement, Volume 2 is an exploration of key trends impacting the futures of public engagement and possible future scenarios. These scenarios were developed using a collective scenario process, which Volume 3 describes in detail, in the hopes that foresight and public engagement practitioners might find use in iterating and utilizing this process to explore participatory future-focused conversations

Pirellulosomes: a new type of membrane-bounded cell compartment in planctomycete bacteria of the genus Pirellula

A distinct type of cellular organization was found in two species of the planctomycete genus Pirellula, Pirellula marina and Pirellula staleyi. Both species possess two distinct regions within the cell which are separated by a single membrane. The major region of the cell, the pirellulosome, contains the fibrillar condensed nucleoid. The other area, the polar cap region, forms a continuous layer surrounding the entire pirellulosome and displays a cap of asymmetrically distributed material at one cell pole. Immuno- and cytochemical-labelling of P. marina demonstrated that DNA is located exclusively within the pirellulosome; cell RNA is concentrated in the pirellulosome, with some RNA also located in the polar cap region

Estimating and exploring the proportions of inter- and intrastate cattle shipments in the United States

Mathematical models are key tools for the development of surveillance, preparedness and response plans for the potential events of emerging and introduced foreign animal diseases. Creating these types of plans requires data; when data are incomplete, mathematical models can help fill in missing information, provided they are informed by the data that are available. In the United States, the most complete national-scale data available on cattle shipments are based on Interstate Certificates of Veterinary Inspection, which track the shipment of cattle between states; data on intrastate cattle shipments are lacking. Here we develop four new datasets on intrastate cattle shipments in the U.S., including an expert elicitation survey covering 19 states and territories and three state-level brand inspection data sets. The expert elicitation survey provides estimates on the proportion of shipments that travel interstate over multiple regions of the U.S. These survey data also identify differences in shipment patterns between regions, cattle commodity types, and sectors of the cattle industry. These survey data cover more states than any other source of intrastate data; however, one limitation of these data is the small number of participating experts in many of the states, only seven of the 19 responding states and territories had a group size of three or larger. The brand data sets include origin and destination information for both intra- and interstate shipments. These data, therefore, also provide detailed information on the proportion of interstate shipments in three Western states, including the temporal and geographic variation in shipments. Because the survey and brand data overlap in the Western U.S., they can be compared. We find that in the Western U.S. the expert estimates of the overall proportion of cattle shipments matched the brand data well. However, the experts estimated that there would be larger differences in beef and dairy shipments than the brand data show. This suggests the cattle industries in the West may be sending similar proportions of commodity specific cattle shipments over state lines. We additionally used the expert survey data to explore how differences in the proportion of interstate shipments can change predictions about cattle shipment patterns using the example of model-guided suggestions for targeted surveillance in Texas. Together these four data sets are the most extensive and geographically comprehensive information to date on intrastate cattle shipments. Additionally, our analyses on predicted shipment patterns suggest that assumptions about intrastate shipments could have consequences for targeted surveillance

Estimating and exploring the proportions of inter- and intrastate cattle shipments in the United States

Mathematical models are key tools for the development of surveillance, preparedness and response plans for the potential events of emerging and introduced foreign animal diseases. Creating these types of plans requires data; when data are incomplete, mathematical models can help fill in missing information, provided they are informed by the data that are available. In the United States, the most complete national-scale data available on cattle shipments are based on Interstate Certificates of Veterinary Inspection, which track the shipment of cattle between states; data on intrastate cattle shipments are lacking. Here we develop four new datasets on intrastate cattle shipments in the U.S., including an expert elicitation survey covering 19 states and territories and three state-level brand inspection data sets. The expert elicitation survey provides estimates on the proportion of shipments that travel interstate over multiple regions of the U.S. These survey data also identify differences in shipment patterns between regions, cattle commodity types, and sectors of the cattle industry. These survey data cover more states than any other source of intrastate data; however, one limitation of these data is the small number of participating experts in many of the states, only seven of the 19 responding states and territories had a group size of three or larger. The brand data sets include origin and destination information for both intra- and interstate shipments. These data, therefore, also provide detailed information on the proportion of interstate shipments in three Western states, including the temporal and geographic variation in shipments. Because the survey and brand data overlap in the Western U.S., they can be compared. We find that in the Western U.S. the expert estimates of the overall proportion of cattle shipments matched the brand data well. However, the experts estimated that there would be larger differences in beef and dairy shipments than the brand data show. This suggests the cattle industries in the West may be sending similar proportions of commodity specific cattle shipments over state lines. We additionally used the expert survey data to explore how differences in the proportion of interstate shipments can change predictions about cattle shipment patterns using the example of model-guided suggestions for targeted surveillance in Texas. Together these four data sets are the most extensive and geographically comprehensive information to date on intrastate cattle shipments. Additionally, our analyses on predicted shipment patterns suggest that assumptions about intrastate shipments could have consequences for targeted surveillance

NF-κB/mTOR/MYC Axis Drives PRMT5 Protein Induction After T Cell Activation via Transcriptional and Non-transcriptional Mechanisms

Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) mediated by CD4+ T cells and modeled via experimental autoimmune encephalomyelitis (EAE). Inhibition of PRMT5, the major Type II arginine methyltransferase, suppresses pathogenic T cell responses and EAE. PRMT5 is transiently induced in proliferating memory inflammatory Th1 cells and during EAE. However, the mechanisms driving PRMT5 protein induction and repression as T cells expand and return to resting is currently unknown. Here, we used naive mouse and memory mouse and human Th1/Th2 cells as models to identify mechanisms controlling PRMT5 protein expression in initial and recall T cell activation. Initial activation of naive mouse T cells resulted in NF-κB-dependent transient Prmt5 transcription and NF-κB, mTOR and MYC-dependent PRMT5 protein induction. In murine memory Th cells, transcription and miRNA loss supported PRMT5 induction to a lesser extent than in naive T cells. In contrast, NF-κB/MYC/mTOR-dependent non-transcriptional PRMT5 induction played a major role. These results highlight the importance of the NF-κB/mTOR/MYC axis in PRMT5-driven pathogenic T cell expansion and may guide targeted therapeutic strategies for MS

326 The anti-TIGIT antibody M6223 induces significant anti-tumor efficacy and immune response via multiple mechanisms of action

BackgroundM6223 is a fully human antagonistic anti-T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) antibody in IgG1 format with Fc-mediated effector function.MethodsThe ability of M6223 to block the interaction of TIGIT with its ligands, CD155 and CD112, and the interaction of TIGIT with CD226 was determined by a flow cytometry-based binding assay. The anti-tumor efficacy, immune profile, and effector function of M6223 were investigated in syngeneic tumor models in huTIGIT knock-in mice. M6223 was either formatted with an effector competent mouse IgG2c constant region (M6223-muIgG2c) or formatted with effector null mouse IgG1-D256A constant region (M6223-muIgG1) as two versions of chimeric antibodies for the in vivo studies.ResultsM6223 dose-dependently blocked the binding of TIGIT to its ligands, including CD155 and CD112, thereby inhibiting a TIGIT-mediated immunosuppressive pathway. In addition, M6223 interrupted the interaction of TIGIT with the costimulatory receptor CD226. By blocking the interactions, the chimeric protein M6223-muIgG2c showed anti-tumor efficacy in multiple tumor models, including an MC38 tumor model (figure 1), and generated tumor antigen-specific long-term protective immunity in immunocompetent huTIGIT knock-in mice. M6223 monotherapy dose-dependently elevated the ratio of CD8+ cytotoxic T cells to regulatory T cells and the ratio of CD226 to TIGIT expression in immune cells in the tumor microenvironment. We also found that M6223 selectively depleted suppressive and exhausted TIGIT+ immune cell subsets and the anti-tumor activity of effector null M6223-muIgG1 was significantly lost (p<0.0001), suggesting that Fc-mediated effector function contributes to M6223 anti-tumor activity. Antibody depletion studies demonstrated that CD8+ T cells and natural killer cells contributed to the anti-tumor activity of M6223 in a complementary manner.Abstract 326 Figure 1M6223-muIgG2c displayed dose-dependent anti-tumor efficacy. M6223-muIgG2c displayed dose-dependent anti-tumor efficacy in an MC38 tumor model in hTIGIT knock-in mice.ConclusionsGiven that TIGIT blockade can inhibit an immunosuppressive pathway as well as remove the suppression on a costimulatory pathway, M6223 has the potential to induce an anti-tumor immune response by three complementary mechanisms: direct blockade of the TIGIT pathway, stimulation of CD226 dimerization/activation, and depletion of TIGIT+ immune subsets by Fc-mediated effector function. Our data demonstrate that these complementary mechanisms orchestrate the anti-tumor activity of M6223. A Phase I, first-in-human clinical trial (NCT04457778) is underway to determine the safety, tolerability, maximum tolerated dose and recommended dose for expansion of M6223 as a single agent (Part 1A) and in combination with bintrafusp alfa (Part 1B) in patients with metastatic or locally advanced solid unresectable tumors.Ethics ApprovalAll animal experiments were performed in accordance with EMD Serono Research & Development Institute, Inc., Billerica, MA, USA, an affiliate of Merck KGaA (protocol 17-008, 20-005) and Wuxi AppTec Animal Care and Use Committee (IACUC) guidelines

Prediction Space Weather Using an Asymmetric Cone Model for Halo CMEs

Halo coronal mass ejections (HCMEs) are responsible of the most severe geomagnetic storms. A prediction of their geoeffectiveness and travel time to Earth's vicinity is crucial to forecast space weather. Unfortunately coronagraphic observations are subjected to projection effects and do not provide true characteristics of CMEs. Recently, Michalek (2006, {\it Solar Phys.}, {\bf237}, 101) developed an asymmetric cone model to obtain the space speed, width and source location of HCMEs. We applied this technique to obtain the parameters of all front-sided HCMEs observed by the SOHO/LASCO experiment during a period from the beginning of 2001 until the end of 2002 (solar cycle 23). These parameters were applied for the space weather forecast. Our study determined that the space speeds are strongly correlated with the travel times of HCMEs within Earth's vicinity and with the magnitudes related to geomagnetic disturbances