Mathematical model of a three-stage innate immune response to a pneumococcal lung infection

ManuscriptPneumococcal pneumonia is a leading cause of death and a major source of human morbidity. The initial immune response plays a central role in determining the course and outcome of pneumococcal disease. We combine bacterial titer measurements from mice infected with Streptococcus pneumoniae with mathematical modeling to investigate the coordination of immune responses and the effects of initial inoculum on outcome. To evaluate the contributions of individual components, we systematically build a mathematical model from three subsystems that describe the succession of defensive cells in the lung: resident alveolar macrophages, neutrophils and monocyte-derived macrophages. The alveolar macrophage response, which can be modeled by a single differential equation, can by itself rapidly clear small initial numbers of pneumococci. Extending the model to include the neutrophil response required additional equations for recruitment cytokines and host cell status and damage. With these dynamics, two outcomes can be predicted: bacterial clearance or sustained bacterial growth. Finally, a model including monocyte-derived macrophage recruitment by neutrophils suggests that sustained bacterial growth is possible even in their presence. Our model quantifies the contributions of cytotoxicity and immune-mediated damage in pneumococcal pathogenesis

TBK1 and IKKε act redundantly to mediate STING-induced NF-κB responses in myeloid cells

Stimulator of Interferon Genes (STING) is a critical component of host innate immune defense but can contribute to chronic autoimmune or autoinflammatory disease. Once activated, the cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS)-STING pathway induces both type I interferon (IFN) expression and nuclear factor-κB (NF-κB)-mediated cytokine production. Currently, these two signaling arms are thought to be mediated by a single upstream kinase, TANK-binding kinase 1 (TBK1). Here, using genetic and pharmacological approaches, we show that TBK1 alone is dispensable for STING-induced NF-κB responses in human and mouse immune cells, as well as in vivo. We further demonstrate that TBK1 acts redundantly with IκB kinase ε (IKKε) to drive NF-κB upon STING activation. Interestingly, we show that activation of IFN regulatory factor 3 (IRF3) is highly dependent on TBK1 kinase activity, whereas NF-κB is significantly less sensitive to TBK1/IKKε kinase inhibition. Our work redefines signaling events downstream of cGAS-STING. Our findings further suggest that cGAS-STING will need to be targeted directly to effectively ameliorate the inflammation underpinning disorders associated with STING hyperactivity

The Exosome Component Rrp6 Is Required for RNA Polymerase II Termination at Specific Targets of the Nrd1-Nab3 Pathway

Publisher’s version made available under a Creative Commons license.The exosome and its nuclear specific subunit Rrp6 form a 3'-5' exonuclease complex that regulates diverse aspects of RNA biology including 3' end processing and degradation of a variety of noncoding RNAs (ncRNAs) and unstable transcripts. Known targets of the nuclear exosome include short (<1000 bp) RNAPII transcripts such as small noncoding RNAs (snRNAs), cryptic unstable transcripts (CUTs), and some stable unannotated transcripts (SUTs) that are terminated by an Nrd1, Nab3, and Sen1 (NNS) dependent mechanism. NNS-dependent termination is coupled to RNA 3' end processing and/or degradation by the Rrp6/exosome in yeast. Recent work suggests Nrd1 is necessary for transcriptome surveillance, regulating promoter directionality and suppressing antisense transcription independently of, or prior to, Rrp6 activity. It remains unclear whether Rrp6 is directly involved in termination; however, Rrp6 has been implicated in the 3' end processing and degradation of ncRNA transcripts including CUTs. To determine the role of Rrp6 in NNS termination globally, we performed RNA sequencing (RNA-Seq) on total RNA and perform ChIP-exo analysis of RNA Polymerase II (RNAPII) localization. Deletion of RRP6 promotes hyper-elongation of multiple NNS-dependent transcripts resulting from both improperly processed 3' RNA ends and faulty transcript termination at specific target genes. The defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of depleting Nrd1 from the nucleus. Elongated transcripts were identified within all classes of known NNS targets with the largest changes in transcription termination occurring at CUTs. Interestingly, the extended transcripts that we have detected in our studies show remarkable similarity to Nrd1-unterminated transcripts at many locations, suggesting that Rrp6 acts with the NNS complex globally to promote transcription termination in addition to 3' end RNA processing and/or degradation at specific targets

Functional screening of amplification outlier oncogenes in organoid models of early tumorigenesis

Genomics; Organoid; Squamous cancerGenómica funcional; Organoide; Cáncer escamosoGenòmica funcional; Organoide; Càncer escamósSomatic copy number gains are pervasive across cancer types, yet their roles in oncogenesis are insufficiently evaluated. This inadequacy is partly due to copy gains spanning large chromosomal regions, obscuring causal loci. Here, we employed organoid modeling to evaluate candidate oncogenic loci identified via integrative computational analysis of extreme copy gains overlapping with extreme expression dysregulation in The Cancer Genome Atlas. Subsets of “outlier” candidates were contextually screened as tissue-specific cDNA lentiviral libraries within cognate esophagus, oral cavity, colon, stomach, pancreas, and lung organoids bearing initial oncogenic mutations. Iterative analysis nominated the kinase DYRK2 at 12q15 as an amplified head and neck squamous carcinoma oncogene in p53−/− oral mucosal organoids. Similarly, FGF3, amplified at 11q13 in 41% of esophageal squamous carcinomas, promoted p53−/− esophageal organoid growth reversible by small molecule and soluble receptor antagonism of FGFRs. Our studies establish organoid-based contextual screening of candidate genomic drivers, enabling functional evaluation during early tumorigenesis.This work was supported by the NCI Cancer Target Discovery and Development (CTD∧2) Network (U01CA217851, C.J.K and C.C.; U01CA176058, W.C.H.). Support was also provided by NIH K08DE027730 and D.R. discretionary funds to A.A.S., AEI RYC2019- 026576-I, “LaCaixa” Foundation LCF/PR/PR17/51120011 to J.A.S., and NIH U54CA224081, NIH U01CA199241, Emerson Collective, Ludwig Cancer Research, and Stand Up To Cancer to C.J.K. This manuscript is dedicated to the memories of Dr. Daniela Gerhard and Dr. Kenneth Scott

A controlled study of virtual reality in first-year magnetostatics

Stereoscopic virtual reality (VR) has experienced a resurgence due to flagship products such as the Oculus Rift, HTC Vive and smartphone-based VR solutions like Google Cardboard. This is causing the question to resurface: how can stereoscopic VR be useful in instruction, if at all, and what are the pedagogical best practices for its use? To address this, and to continue our work in this sphere, we performed a study of 289 introductory physics students who were sorted into three different treatment types: stereoscopic virtual reality, WebGL simulation, and static 2D images, each designed to provide information about magnetic fields and forces. Students were assessed using preliminary items designed to focus on heavily-3D systems. We report on assessment reliability, and on student performance. Overall, we find that students who used VR did not significantly outperform students using other treatment types. There were significant differences between sexes, as other studies have noted. Dependence on students' self-reported 3D videogame play was observed, in keeping with previous studies, but this dependence was not restricted to the VR treatment.Comment: 6 pages, 4 figures, submitted to the 2019 Physics Education Research Conferenc

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation

In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes

Effect of 1918 PB1-F2 Expression on Influenza A Virus Infection Kinetics

Relatively little is known about the viral factors contributing to the lethality of the 1918 pandemic, although its unparalleled virulence was likely due in part to the newly discovered PB1-F2 protein. This protein, while unnecessary for replication, increases apoptosis in monocytes, alters viral polymerase activity in vitro, enhances inflammation and increases secondary pneumonia in vivo. However, the effects the PB1-F2 protein have in vivo remain unclear. To address the mechanisms involved, we intranasally infected groups of mice with either influenza A virus PR8 or a genetically engineered virus that expresses the 1918 PB1-F2 protein on a PR8 background, PR8-PB1-F2(1918). Mice inoculated with PR8 had viral concentrations peaking at 72 hours, while those infected with PR8-PB1-F2(1918) reached peak concentrations earlier, 48 hours. Mice given PR8-PB1-F2(1918) also showed a faster decline in viral loads. We fit a mathematical model to these data to estimate parameter values. The model supports a higher viral production rate per cell and a higher infected cell death rate with the PR8-PB1-F2(1918) virus. We discuss the implications these mechanisms have during an infection with a virus expressing a virulent PB1-F2 on the possibility of a pandemic and on the importance of antiviral treatments

Copper (I) SNS Pincer Complexes: Impact of Ligand Design and Solvent Coordination on Conformer Interconversion from Spectroscopic and Computational Studies

The syntheses and detailed characterizations (X-ray crystallography, NMR spectroscopy, cyclic voltammetry, infrared spectroscopy, electrospray mass spectrometry, and elemental analyses) of two new Cu(I) pincer complexes are reported. The pincer ligand coordinates through one nitrogen and two sulfur donor atoms and is based on bis-imidazole or bis-triazole precursors. These tridentate SNS ligands incorporate pyridine and thione-substituted imidazole or triazole functionalities with connecting methylene units that provide flexibility to the ligand backbone and enable high bite-angle binding. Variable temperature 1H NMR analysis of these complexes and of a similar zinc(II) SNS system shows that all are fluxional in solution and permits the determination of ΔGexp‡ and ΔSexp‡. DFT calculations are used to model the fluxionality of these complexes and indicate that a coordinating solvent molecule can promote hemilability of the SNS ligand by lowering the energy barrier involved in the partial rotation of the methylene units