A systems and molecular analysis of G protein-mediated signalling

The ability of cells to respond correctly to signals from their microenvironment is an essential prerequisite of life. Many external signals are detected through G protein-coupled receptor (GPCR) signalling pathways, which control all aspects of eukaryotic physiology. Ligand-bound GPCRs initiate signalling by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins, thereby facilitating activation of downstream effectors. Signalling is terminated by the hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, in a reaction catalysed by the regulator of G protein signalling (RGS) proteins. Due to the problem of complexity in higher eukaryotic GPCR signalling, the matingresponse in Schizosaccharomyces pombe has been used to study GPCR signalling in isolation. In vivo data from quantitative assays of reporter strains and live-cell uorescence microscopy informs the development of an ordinary differential equation model of the signalling pathway, first described by Smith et al., 2009. The rate of nucleotide exchange on the Gα (Gpa1) is a key molecular mechanism controlling duration and amplitude of signalling response. The in uence of this is investigated through characterisation of Gpa1 nucleotide exchange mutants and perturbation of reaction rate parameters in the computational model. Further, this thesis also presents data relating to the temporal and spatial regulation of Rgs1 (the sole RGS protein for Gpa1). Using an inter-disciplinary approach, evidence is provided to suggest that an interaction between Rgs1 and the C-terminal tail of the GPCR (Mam2) tethers Rgs1 to the plasma membrane to facilitate its function. Finally, quantification of signalling at the single cell level is described. Time-lapse livecell imaging of uorescent reporter cells is optimised and single cell signalling response quantified using image analysis software. Single cell quantification provides greater insight into temporal dynamics, cell-to-cell variability, and highlights the existence of mechanisms for cellular decision-making

Cellular mRNAs access second ORFs using a novel amino acid sequence-dependent coupled translation termination-reinitiation mechanism

Polycistronic transcripts are considered rare in the human genome. Initiation of translation of internal ORFs of eukaryotic genes has been shown to use either leaky scanning or highly structured IRES regions to access initiation codons. Studies on mammalian viruses identified a mechanism of coupled translation termination-reinitiation that allows translation of an additional ORF. Here, the ribosome terminating translation of ORF-1 translocates upstream to reinitiate translation of ORF-2. We have devised an algorithm to identify mRNAs in the human transcriptome in which the major ORF-1 overlaps a second ORF capable of encoding a product of at least 50 aa in length. This identified 4368 transcripts representing 2214 genes. We investigated 24 transcripts, 22 of which were shown to express a protein from ORF-2 highlighting that 3' UTRs contain protein-coding potential more frequently than previously suspected. Five transcripts accessed ORF-2 using a process of coupled translation termination-reinitiation. Analysis of one transcript, encoding the CASQ2 protein, showed that the mechanism by which the coupling process of the cellular mRNAs was achieved was novel. This process was not directed by the mRNA sequence but required an aspartate-rich repeat region at the carboxyl terminus of the terminating ORF-1 protein. Introduction of wobble mutations for the aspartate codon had no effect, whereas replacing aspartate for glutamate repeats eliminated translational coupling. This is the first description of a coordinated expression of two proteins from cellular mRNAs using a coupled translation termination-reinitiation process and is the first example of such a process being determined at the amino acid level

Probabilistic encoding of stimulus strength in astrocyte global calcium signals

Astrocyte calcium signals can range in size from subcellular microdomains to waves that spread through the whole cell (and into connected cells). The differential roles of such local or global calcium signaling are under intense investigation, but the mechanisms by which local signals evolve into global signals in astrocytes are not well understood, nor are the computational rules by which physiological stimuli are transduced into a global signal. To investigate these questions, we transiently applied receptor agonists linked to calcium signaling to primary cultures of cerebellar astrocytes. Astrocytes repetitively tested with the same stimulus responded with global signals intermittently, indicating that each stimulus had a defined probability for triggering a response. The response probability varied between agonists, increased with agonist concentration, and could be positively and negatively modulated by crosstalk with other signaling pathways. To better understand the processes determining the evolution of a global signal, we recorded subcellular calcium “puffs” throughout the whole cell during stimulation. The key requirement for puffs to trigger a global calcium wave following receptor activation appeared to be the synchronous release of calcium from three or more sites, rather than an increasing calcium load accumulating in the cytosol due to increased puff size, amplitude, or frequency. These results suggest that the concentration of transient stimuli will be encoded into a probability of generating a global calcium response, determined by the likelihood of synchronous release from multiple subcellular sites

The single cell transcriptional landscape of esophageal adenocarcinoma and its modulation by neoadjuvant chemotherapy

Immune checkpoint blockade has recently proven effective in subsets of patients with esophageal adenocarcinoma (EAC) but little is known regarding the EAC immune microenvironment. We determined the single cell transcriptional profile of EAC in 8 patients who were treatment-naive (n = 4) or had received neoadjuvant chemotherapy (n = 4). Analysis of 52,387 cells revealed 10 major cell subsets of tumor, immune and stromal cells. Prior to chemotherapy tumors were heavy infiltrated by T regulatory cells and exhausted effector T cells whilst plasmacytoid dendritic cells were markedly expanded. Two dominant cancer-associated fibroblast populations were also observed whilst endothelial populations were suppressed. Pathological remission following chemotherapy associated with broad reversal of immune abnormalities together with fibroblast transition and an increase in endothelial cells whilst a chemoresistant epithelial stem cell population correlated with poor response. These findings reveal features that underlie and limit the response to current immunotherapy and identify a range of novel opportunities for targeted therapy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12943-022-01666-x

Plasticity of Neuron-Glial Transmission: Equipping Glia for Long-Term Integration of Network Activity

The capacity of synaptic networks to express activity-dependent changes in strength and connectivity is essential for learning and memory processes. In recent years, glial cells (most notably astrocytes) have been recognized as active participants in the modulation of synaptic transmission and synaptic plasticity, implicating these electrically nonexcitable cells in information processing in the brain. While the concept of bidirectional communication between neurons and glia and the mechanisms by which gliotransmission can modulate neuronal function are well established, less attention has been focussed on the computational potential of neuron-glial transmission itself. In particular, whether neuron-glial transmission is itself subject to activity-dependent plasticity and what the computational properties of such plasticity might be has not been explored in detail. In this review, we summarize current examples of plasticity in neuron-glial transmission, in many brain regions and neurotransmitter pathways. We argue that induction of glial plasticity typically requires repetitive neuronal firing over long time periods (minutes-hours) rather than the short-lived, stereotyped trigger typical of canonical long-term potentiation. We speculate that this equips glia with a mechanism for monitoring average firing rates in the synaptic network, which is suited to the longer term roles proposed for astrocytes in neurophysiology. Plasticity as the Cellular Basis of Learning and Memory in the Central Nervous System At a high level of abstraction, the brain is essentially an organ that detects environmental stimuli, processes the received sensory information, and initiates an appropriate motor response. From this perspective, the primary role of the brain is information processing, and the computational processes associated with transforming input to output are centred on the network of trillions of synapses through which the signals are relayed. The train of action potentials initiated in sensory neurons must be transduced by the central synaptic networks in such a way as to reliably trigger a pattern of action potentials in the motor neurons that effect the necessary coordinated activation of muscles needed to evoke a behavioural response. It is thus widely accepted that, despite defying human comprehension, there must be a particular spatiotemporal pattern of network activity reliably associated with generating a given response to a given external cue. To cope with a complex and changing environment, the synaptic network must also be adaptable, such that experience can refine and reorganize the spatiotemporal patterns of network activity in response to, for example, injurious stimuli. This adaptability requires controlled alteration of synaptic strength, a phenomenon termed synaptic plasticity The forms and mechanisms of synaptic plasticity have been extensively studied for many decades in many brain region

Measuring the complexity of social associations using mixture models

We propose a method for examining and measuring the complexity of animal social networks that are characterized using association indices. The method focuses on the diversity of types of dyadic relationship within the social network. Binomial mixture models cluster dyadic relationships into relationship types, and variation in the preponderance and strength of these relationship types can be used to estimate association complexity using Shannon’s information index. We use simulated data to test the method, and find that models chosen using integrated complete likelihood give estimates of complexity that closely reflect the true complexity of social systems, but these estimates can be downwardly biased by low intensity sampling and upwardly biased by extreme overdispersion within components. We also illustrate the use of the method on two real data sets. The method could be extended for use on interaction rate data using Poisson mixture models, or on multidimensional relationship data using multivariate mixture models

Cosmological Limits on the Neutrino Mass from the Lya Forest

The Lya forest in quasar spectra probes scales where massive neutrinos can strongly suppress the growth of mass fluctuations. Using hydrodynamic simulations with massive neutrinos, we successfully test techniques developed to measure the mass power spectrum from the forest. A recent observational measurement in conjunction with a conservative implementation of other cosmological constraints places upper limits on the neutrino mass: m_nu < 5.5 eV for all values of Omega_m, and m_nu < 2.4 (Omega_m/0.17 -1) eV, if 0.2 < Omega_m <0.5 as currently observationally favored (both 95 % C.L.).Comment: 4 pages, 2 ps figures, REVTex, submitted to Phys. Rev. Let

Trends in the Use, Sociodemographic Correlates, and Undertreatment of Prescription Medications for Chronic Obstructive Pulmonary Disease among Adults with Chronic Obstructive Pulmonary Disease in the United States from 1999 to 2010

BACKGROUND: The extent to which patients with COPD are receiving indicated treatment with medications to improve lung function and recent trends in the use of these medications is not well documented in the United States. The objective of this study was to examine trends in prescription medications for COPD among adults in the United States from 1999 to 2010. METHODS: We performed a trend analysis using data from up to 1426 participants aged ≥20 years with self-reported COPD from six national surveys (National Health and Nutrition Examination Survey 1999-2010). RESULTS: During 2009-2010, the age-adjusted percentage of participants who used any kind of medication was 44.2%. Also during 2009-2010, the most commonly used medications were short-acting agents (36.0%), inhaled corticosteroids (ICS) (18.3%), and LABAs (16.7%). The use of long-acting beta-2 agonists (LABAs) (p for trend CONCLUSION: The percentages of adults with COPD who reported having various classes of prescription medications that improve airflow limitations changed markedly from 1999-2000 to 2009-2010. However, many adults with COPD did not report having recommended prescription medications