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Carbon dioxide detection in biological systems
Theo Murphy international scientific meeting organised by Professor Martin Cann, Dr Vicki Linthwaite and Dr Eoin Cummins.
This meeting will unite researchers from the plant and animal kingdoms to discuss the common goal of understanding the molecular basis of carbon dioxide detection.
Carbon dioxide is essential for life on earth. It is central to physiological processes including photosynthesis, metabolism, homeostasis, chemosensing and pathogenesis. Given recent and rapid developments in our understanding of mechanisms that underpin carbon dioxide detection in both the plant and animal kingdoms, a new forum must be developed to bring together researchers in these fields.
This forum will identify common themes in carbon dioxide detection across different biological systems and exchange methodologies that can be bought to bear on different biological systems. Furthermore, its members will identify new approaches that can benefit our identification of carbon dioxide detection mechanisms across diverse species. The forum will foster the creation of new scientific community/network that will enhance the pursuit of knowledge in this area and form the foundations for future collaborative publications and funding proposals.
Speaker abstracts will be available closer to the meeting. Recorded audio of the presentations will be available on this page after the meeting has taken place.
Meeting papers will be published in a future issue of Interface Focus.
Poster session
There will be a poster session at 17:00 on Monday 2 December 2019. If you would like to apply to present a poster please submit your proposed title, abstract (not more than 200 words and must be in third person), author list, name of the proposed presenter and institution to the Scientific Programmes team with the subject heading "Carbon Dioxide: poster abstract" no later than Friday 18 October 2019.
Please note that places are limited and posters are selected at the scientific organisers' discretion. Poster abstracts will only be considered if the presenter is registered to attend the meeting.
Attending this event
This is a residential conference, which allows for increased discussion and networking.
- Free to attend
- Limited places
- Please request a registration invitation above
- Catering and accommodation available to purchase during registration
Enquiries: contact the Scientific Programmes team
Organisers
Schedule
Chair
Professor Martin Cann, Durham University, UK
Professor Martin Cann, Durham University, UK
Martin Cann did his PhD in Molecular Virology at the University of Reading and studied cyclic nucleotide signalling as a PDRA with Lonny Levin (Weill Medical College of Cornell University) and Dave Garbers (HHMI, University of Texas Southwestern). He was part of the team (with Lonny Levin) that identified the soluble adenylyl cyclase as a carbon-activated signalling molecule in mammals. In his own laboratory, at Durham University, he has had an interest in the general phenomenon of regulation of the cAMP signalling pathway by inorganic carbon. More recently he has been interested in carbon dioxide-mediated post-translational modifications and has established methodology to identify carbon dioxide-binding sites on protein.
| 09:00 - 09:05 | Welcome by the Royal Society and organisers |
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| 09:05 - 09:35 |
Protein carbamylation: the discovery of a carbon dioxide control system
Carbon dioxide (CO2) is fundamental to life with critical roles in respiration, photosynthesis and acid-base homeostasis. Carbamates are formed rapidly but reversibly by the nucleophilic attack of an uncharged amine on CO2 at physiological temperatures and pressures. The presence of this post-translational modification has been demonstrated in a small number of key proteins, such as RuBisCO and haemoglobin. The systematic identification of carbamates has been hindered due to their labile nature, therefore previous work has involved their study under non-physiological conditions. We have developed a novel technology using a chemical trapping technique to covalently modify carbamates and remove their labile nature1. This has been combined with downstream proteomic analysis via tryptic digest and ESI-MS to validate the carbamate modifications identified. Our method successfully identified the haemoglobin carbamate binding site under physiologically relevant conditions. These results were confirmed using ESI-MS combined with 12C and 13C isotope incorporation. This research has produced a method that utilises a pre-existing chemical reagent to remove the labile nature of carbamates and thereby provides the first description of systematic carbamate identification in a physiologically relevant environment. Screening of mammalian protein lysate identified two sites on the ubiquitin protein which were able to bind CO2. Carbamylation of ubiquitin has been demonstrated directly to influence the protein ubiquitin cross linking at this site in vitro and in cellulo. We propose ubiquitin as a universal target able to mediate the diverse cellular affects of CO2. 
Dr Vicki Linthwaite, Durham University, UK
Dr Vicki Linthwaite, Durham University, UKVicki completed her BSc in Biochemistry and MSc in Bioscience Technologies from the University of York. She completed her PhD on the development of a technology for trapping protein carbamates with Professor Martin Cann from Durham University in 2017. She has since been carrying out postdoctoral studies within Durham University investigating the functional role of carbamylation within a cellular environment. |
| 09:35 - 09:45 | Discussion |
| 09:45 - 10:15 |
Stomatal responses to carbon dioxide
In response to elevated concentrations of carbon dioxide the pores on the surfaces of leaves known as stomata close. Closure restricts the loss of water vapour from the plant and the uptake of CO2 from the atmosphere.The stomatal pore is surrounded by two specialised cells known as guard cells. When guard cells lose turgor the stomatal pore closes.Research over the past 20 years has begun to identify the elements of the intracellular signal transduction pathway responsible for coupling CO2 perception with reductions in guard cell turgor. The picture emerging is that some signalling components are shared with the signalling pathways used by other closure-inducing signals while others are restricted to the CO2 response. This lecture will focus on recently identified components and on the role of the closure-inducing plant hormone abscisic acid in the guard cell CO2 response. 
Professor Alistair Hetherington, Bristol University, UK
Professor Alistair Hetherington, Bristol University, UKAlistair Hetherington has worked in the University of Bristol, UK since 2006 where he holds the Melville Wills Chair of Botany. He is a graduate (BSc, PhD) of the University of St Andrews and before moving to Bristol worked at the Universities of Edinburgh and Lancaster. He has also held visiting Fellowships at the University of Oxford (St Catherine's and Magdalen). His primary interest is to understand signal transduction systems and to do this he uses the stomatal guard cell as a model. Originally his focus was on calcium-based intracellular signalling but his interests now extend to understanding how extracellular signals are perceived and integrated in guard cells. He has worked on how CO2 controls stomatal development and the aperture of the stomatal pore. |
| 10:15 - 10:25 | Discussion |
| 10:25 - 10:55 | Coffee |
| 10:55 - 11:25 |
Therapeutically targeting CO<sub>2</sub>/HCO<sub>3</sub>/pH sensing soluble adenylyl cyclase
Signaling via the prototypical second messenger cyclic AMP (cAMP) is compartmentalized; it mediates its various responses via multiple, independently regulated cAMP signaling microdomains. Cyclic AMP is produced by adenylyl cyclases (ACs), and in mammalian cells, there are two distinct families of ACs; G protein regulated transmembrane adenylyl cyclases (tmAC) and soluble adenylyl cyclase (sAC). TmACs anchor cAMP signaling microdomains at the plasma membrane, where they respond to hormonal signals operating via G protein coupled receptors. In contrast, sAC can be found distributed throughout the cytoplasm and inside cellular organelles where it defines multiple, independently regulated intracellular cAMP signaling microdomains. sAC is also biochemically distinct from tmACs; sAC activity is uniquely and directly regulated by bicarbonate (HCO3-) ions. Due to the ubiquitous presence of carbonic anhydrases (CA), which catalyze the instantaneous equilibration of carbon dioxide (CO2), HCO3-, and protons, mammalian sAC, and its HCO3--regulated orthologs throughout the kingdoms of life, serve as Nature’s physiological CO2/HCO3-/pHi sensors. The mechanism of bicarbonate regulation of sAC, along with recent advances in pharmacological modulators, including potential novel therapeutic uses, will be discussed. 
Professor Lonny Levin, Weill Medical College of Cornell University, USA
Professor Lonny Levin, Weill Medical College of Cornell University, USADr Levin received his PhD from Cold Spring Harbor laboratory working with Mark Zoller using genetics in yeast to probe the interaction between regulatory and catalytic subunits of the cAMP dependent protein kinase. For post doctoral training, Dr Levin worked with Randall Reed at Johns Hopkins Medical School cloning the Drosophila Rutabaga learning and memory gene encoding Calcium/calmodulin regulated adenylyl cyclase. Since beginning his own laboratory at Weill Cornell Medical College, In collaboration with Dr Jochen Buck, Drs Buck and Levin were the first to purify and clone mammalian soluble adenylyl cyclase (sAC) and demonstrate its regulation by bicarbonate ions. Because bicarbonate is in nearly instantaneous equilibrium with carbon dioxide and intracellular pH due to the ubiquitous presence of carbonic anhydrases, sAC is poised to respond to changes in carbon dioxide production and intracellular pH. |
| 11:25 - 11:35 | Discussion |
| 11:35 - 12:05 |
CO2 sensing and behaviour in insects
Many insects can smell CO2 using specialised seven transmembrane receptors present in olfactory neurons. In the case of blood-feeding insects like mosquitoes, the sensing of a turbulent plume of CO2 indicates a living vertebrate upwind and leads to a series of important behaviours that are critical for host-seeking. First, even a brief turbulent plume of CO2 causes the insect to activate and initiate upwind flight. Second, the flying insect utilizes the plumes of CO2 to navigate upwind towards the source. And third, the exposure to CO2 increases the behavioural attraction to odorants from human skin and to heat and visual stimuli by several fold. Results will be presented on how the conserved transmembrane heteromeric receptor proteins detect CO2, and how it can be modulated by the presence or absence of certain protein subunits. Several other classes of volatile chemicals have been identified that can act as agonists, inverse-agonists or antagonists of the CO2-receptor and showed how they can predictably alter insect behaviour. Machine learning based algorithms have been applied to screen millions of molecules for such activities on the receptor and their utility validated with electrophysiology recordings and behaviour assays. Several lines of evidence have also been uncovered which point to additional roles of the CO2-receptor in detecting other behaviourally relevant odorants and act as one of the central detectors of volatiles for olfactory behaviour in a number of flying insects. 
Professor Anandasankar Ray, University of California, Riverside, USA
Professor Anandasankar Ray, University of California, Riverside, USAAnandasankar Ray is a Professor at University of California, Riverside. He is known for his work on the neurobiology of the olfactory system, using it as a model to understand how to disrupt behavior in mosquitoes that transmit diseases like Malaria, Dengue and Zika. He is also founder of Sensorygen Inc which is developing next generation insect repellents. He also uses the olfactory system to study the role of epigenetic regulation in neuronal development and neuro-degeneration. |
| 12:05 - 12:15 | Discussion |
Chair
Dr Eoin Cummins, University College Dublin, Ireland
Dr Eoin Cummins, University College Dublin, Ireland
Eoin obtained a bachelor’s degree in Pharmacology (2002), and a PhD (2007) from University College Dublin (UCD). His early work focused the role of oxygen sensing hydroxylases in the context of intestinal inflammation. During his post-doctoral career Eoin became interested in the potential role for carbon dioxide as a modulator of inflammatory and immune signaling, in particular in relation to NFkB signaling. The first paper on this work was published in the Journal of Immunology in 2010. Eoin subsequently took up a short-term fellowship in Northwestern University, before returning to UCD and being appointed as an Assistant Professor in Physiology. He currently leads a Science Foundation Ireland funded research group focused on the effects of hypercapnia on inflammation and immunity.
| 13:15 - 13:45 |
CO2 transport
The dogma had been that all gases cross all membranes by dissolving in and diffusing through the lipid phase. Work over the past two decades shows that some membranes have negligible CO2 permeability, and that some membrane proteins—a subset of aquaporins (AQPs) and rhesus (Rh) proteins—can conduct CO2 or other gases. Preliminary data on Xenopus oocytes expressing AQP5 show that CO2 transport through AQP5 increases markedly with injection into the cytosol of small amounts of carbonic anhydrase II (CAII). The approach is to measure alkaline surface pH (pHS) transients during introduction of extracellular CO2/HCO3−. The alkaline pHS spike is greatest early, when the inward CO2 gradient—and thus the extracellular reaction HCO3− + H+ H2O + CO2—is greatest, and wanes as CO2 equilibrates across the cell membrane. Cytosolic CAII promotes the consumption of incoming CO2, thereby maintaining low cytosolic [CO2], maximizing the gradient for CO2 influx, and increasing pHS—far more so in AQP5-expressing than in control oocytes. In additional pHS experiments, the electrogenic Na/HCO3 cotransporter NBCe1 (or electroneutral NBCn1) replaced the AQP5. Earlier preliminary work based on intracellular-pH measurements had suggested that NBCe1 conducts CO2. Now, exposing oocytes expressing NBCe1 (or NBCn1)—without injected CAII—to CO2/HCO3− produces a small transient pHS increase (due to CO2 influx) followed by a large pHS decrease (CO3= uptake). In oocytes expressing NBCe1 or NBCn1, CAII injection markedly accentuates the early transient pHS increase (as for AQP5-expressing oocytes), supporting the hypothesis that NBCe1 and NBCn1 both conduct CO2. 
Professor Walter Boron, Case Western Reserve University, USA
Professor Walter Boron, Case Western Reserve University, USADr Boron is Myers/Scarpa Professor and Chair of Physiology & Biophysics Department at CWRU. He earned his MD and PhD (Physiology & Biophysics) at Washington University, joined Yale as a postdoctoral fellow with Emile Boulpaep in 1978, and remained until 2007, serving as department chair for three 3-year terms. Boron was President of the American Physiological Society, Secretary-General of the International Union of Physiological Sciences, and editor-in-chief of both Physiological Reviews and Physiology. With Boulpaep, he edits the textbook Medical Physiology. Boron developed his interest in acid-base transport/intracellular-pH regulation with PhD mentors Albert Roos and Paul De Weer, and his interest in renal HCO3¯ transport with Boulpaep. His group focuses on three related areas: molecular physiology of the Na+-coupled HCO3¯transporters, molecular CO2/HCO3¯ sensors, and gas channels. Among Boron’s honors are an honorary doctorate from Aarhus University (2014), and election to the National Academy of Medicine (2014). |
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| 13:45 - 13:55 | Discussion |
| 13:55 - 14:25 |
The Calvin Benson cycle – atmospheric CO2 assimilation and prospects for improvement
The photosynthetic carbon reduction (Calvin-Benson) cycle is the primary pathway of atmospheric CO2 assimilation in all photosynthetic organisms. It is the single largest flux of organic carbon in the biosphere, and assimilates about 100 bn tons of carbon a year (15% of the carbon in the atmosphere). Evidence has now accumulated showing that by increasing flow of CO2 through this cycle we can also increase plant yield which has the potential to contribute the future demands of an increasing world population. Analyses of transgenic with altered level of enzymes in the Calvin-Benson cycle has demonstrated the potential for increasing crop productivity through increased rates of photosynthesis and the manipulation of the allocation of photosynthate. This lecture will discuss these approaches and the future potential. 
Professor Christine Raines, Essex University, UK
Professor Christine Raines, Essex University, UKChristine Raines graduated with a BSc (Hons) Agricultural Botany, Glasgow University. Following which she was awarded a Glasgow University Scholarship and studied for a PhD in Photosynthetic electron transport graduating in 1986. Christine’s post-doctoral research started in late 1985 at the Institute of Plant Science Research, Cambridge where she initiated a project cloning nuclear genes encoding enzymes of the C3 cycle. In 1988 Christine was appointed to a faculty position at the University of Essex being promoted to Professor in 2004. Christine was Head of the School of Life Sciences at Essex (2011-2017) and is now Pro-Vice Chancellor Research for the University of Essex. Christine has also held a number of external roles; Editor in Chief, Journal of Experimental Botany (2011- ), Chair of Plant Section, Society of Experimental Biology (2009- ) and SEB President (2017-2019). |
| 14:25 - 14:35 | Discussion |
| 14:35 - 15:00 | Tea |
| 15:00 - 15:30 |
CO2 and lung airway function
Carbon dioxide (CO2), a primary product of oxidative metabolism, can be sensed by eukaryotic cells eliciting specific responses via specific signaling pathways. The physiological and pathophysiological effects of high CO2 conditions (hypercapnia) on the lungs and specific lung cells, which are the primary site of CO2 elimination, are incompletely understood. Dr Shigemura’s group has recently reported using combined unbiased molecular approaches with studies in mice and cell culture systems on the mechanisms by which hypercapnia increased airway smooth muscle contractility. The group described that high CO2 levels cause non-apoptotic caspase-7 activation via the calcium-calpain signal, which cleaves the transcription factor myocyte-specific enhancer factor 2D and in turn downregulates miR-133a that increases RhoA protein abundance and myosin light chain phosphorylation, and thus leads to airway smooth muscle contraction. In demonstration of the clinical relevance of this signaling, the group determined that patients with severe chronic obstructive pulmonary disease (COPD) and hypercapnia had elevated airway resistance, which improved after correction of hypercapnia. These data suggest that hypercapnia is not only a manifestation of severe COPD, but it can also worsen the airflow obstruction. Dr Shigemura will provide a pathophysiological and mechanistic perspective on the effects of hypercapnia on the lung airways and discuss the recent understanding of high CO2 modulation of the airway function. 
Dr Masahiko Shigemura, Northwestern University, USA
Dr Masahiko Shigemura, Northwestern University, USAMasahiko Shigemura is a Research Assistant Professor of Medicine at Northwestern University (USA). He obtained his PhD in Medicine from Hokkaido University (Japan). Afterwards, he carried out post-doctoral research work in the laboratory of Dr Jacob I Sznajder at Northwestern University. During his postdoctoral training, he concentrated on the biology of hypercapnia (an elevation of CO2 in the blood) in lungs and meanwhile, he developed a keen interest in the molecular mechanisms by which hypercapnia activates specific signal transduction pathways in the lung airways. His primary research includes airway smooth muscle contractility during hypercapnia, which is potentially clinically relevant to patients with obstructive lung diseases such as chronic obstructive pulmonary disease (COPD). As a Research Assistant Professor, he has been expanding his scientific repertoire and has been conducting research to address airway pathobiology in hypercapnia. |
| 15:30 - 15:40 | Discussion |
| 15:40 - 16:10 |
CO2 sensing and signal transduction in stomatal guard cells
Plants control CO2 exchange and water loss via stomatal pores. Previous research has suggested that bicarbonate (HCO3-) may directly up-regulate reconstituted SLAC1 channel activity in vitro. However, whether this HCO3- regulation is relevant in planta remains unknown. We have computationally predicted candidate bicarbonate-binding sites within SLAC1 through long-timescale Gaussian-accelerated molecular dynamics (GaMD) simulations. Gas exchange and patch clamp experiments with complemented slac1 mutant plants expressing mutated SLAC1 proteins revealed that in plants one of these SLAC1 residues is specifically required for the stomatal CO2 response, but not for ABA responses. These findings suggest that SLAC1 not only mediates anion efflux from guard cells, but also that SLAC1 could contribute as a CO2/HCO3- sensing in guard cells. These analyses suggest that SLAC1 can function as a “secondary” bicarbonate/CO2 sensor, but not as the primary CO2/bicarbonate sensor. [CO2] elevation and the plant hormone abscisic acid (ABA) both trigger rapid stomatal closure via regulation of ion channels in guard cells. Abscisic acid is known to enhance CO2 responses. However, it has remained unknown whether [CO2]-triggered stomatal closure is directly mediated via activation of the early ABA synthesis and signal transduction pathway and how these pathways converge. To address these questions, stomatal CO2 responses were analyzed in ABA synthesis mutants and ABA receptor mutants. Experiments using higher order mutants show that abscisic acid synthesis and signaling components are essential for robust CO2 responses. Furthermore, direct biochemical and patch clamp analyses of guard cell CO2 and ABA signal transduction were pursued. Moreover, newly developed real-time ABA FRET nano-reporter expressing plants were generated to determine whether CO2 elevation causes rapid ABA concentration changes in guard cells. Taken together these interdisciplinary analyses provide strong evidence for a requirement of basal ABA signal transduction for CO2 signaling and point to a new and unexpected understanding of how CO2 signaling and ABA signaling merge downstream of early ABA and CO2 signaling mechanisms that both close stomata. New findings on CO2 sensing mechanisms and on dissecting CO2-specific signaling in grasses will be presented. A model for early CO2 signal transduction mechanisms that control stomatal movements will be discussed. 
Professor Julian Schroeder, University of Calfornia, San Diego, USA
Professor Julian Schroeder, University of Calfornia, San Diego, USAJulian Schroeder is Novartis Chair and Distinguished Professor at the University of California San Diego. Schroeder identified and characterized of ion channels in higher plants and identified their functions and regulation mechanisms, in particular in stomatal guard cell signal transduction and abiotic stress resistance. His recent research is focused on uncovering the fundamental mechanisms by which plants regulate their stomata in response to drought, abscisic acid and the continuing rise in the atmospheric CO2 concentration. He has received awards, including the Presidential Young Investigator Award (NSF), the ASPB Charles Albert Shull Award (1997), the Blasker Award in Environmental Science and is Churchill Overseas Fellow at Cambridge University. Julian is a member of the U.S. National Academy of Sciences, Fellow of the American Association for the Advancement of Science and member of the German National Academy of Sciences Leopoldina. |
| 16:10 - 16:20 | Discussion |
| 16:20 - 16:50 |
The effects of carbon dioxide on pulmonary inflammatory processes arising from septic and aseptic aetiologies
Acute respiratory distress syndrome (ARDS), a lung disease with rapid onset and high mortality, is characterised by inflammatory signalling pathway activation, infiltration of peripheral leukocytes into the pulmonary space and resultant systemic hypoxia and hypercapnia. Aetiology is typically pathogenic, but also arises due to factors including ventilator induced lung injury (VILI). Permissive or therapeutic hypercapnia are useful anti-inflammatory approaches to ARDS management, but molecular mechanisms are only slowly being revealed. In a series of experiments, rats were buffered renally or ventilated with 5% CO2 and received caecal puncture, E.coli instillation or increased volume ventilation. Assessment was through blood gas analysis, biochemical markers and lung tissue histology. In vitro, lung epithelial and other cells were exposed to increasing CO2 and subject to scratch wound, cytokine, endotoxin or cyclic mechanical stretch injury. Transfection and immunoprecipitation studies examined the influence of CO2 on various points along the NFκB signalling pathway. Hypercapnia attenuated systemic sepsis, though unbuffered alone reduced lung complications. CO2 related benefit was observed in both early and prolonged sepsis. VILI was attenuated by hypercapnia, reflected in a mechanical stretch model of lung epithelium. Hypercapnia diminished the NFκB inflammatory response in multiple cell lines independently of cytoplasmic pH, suggesting direct control by CO2. IKK2 enzymatic activity, IκBα degradation and NFκB translocation were all impaired, as was healing of epithelial scratch wounds, by elevated CO2. In conclusion, hypercapnia is anti-inflammatory in a range of pulmonary injury models, and is inhibitory across many points of the central NFκB inflammatory pathway. 
Professor Daniel O'Toole, National University of Ireland, Galway, Ireland
Professor Daniel O'Toole, National University of Ireland, Galway, IrelandDr Daniel O’Toole graduated in biochemistry at the National University of Ireland, Galway, and following a PhD in Biochemistry at Trinity College Dublin and postdoctoral work in immunology at University College Dublin, returned to Galway as a senior research lecturer in the Discipline of Anaesthesia. His work covers pathological processes and therapeutics for a range of conditions including pneumonia, sepsis and other inflammatory diseases and invokes ventilation, gene and cell therapy approaches. He is particularly interested in carbon dioxide as a potential anti-inflammatory agent in acute respiratory distress syndrome (ARDS). |
| 16:50 - 17:00 | Discussion |
Chair
Dr Vicki Linthwaite, Durham University, UK
Dr Vicki Linthwaite, Durham University, UK
Vicki completed her BSc in Biochemistry and MSc in Bioscience Technologies from the University of York. She completed her PhD on the development of a technology for trapping protein carbamates with Professor Martin Cann from Durham University in 2017. She has since been carrying out postdoctoral studies within Durham University investigating the functional role of carbamylation within a cellular environment.
| 09:00 - 09:30 |
Hypercapnia in the critcally ill
Arterial CO2 tensions (PaCO2) represents a balance between CO2 production and elimination via the lungs, and in health is maintained within a tight range (3.5 – 4.5 kPa). Traditional approaches to CO2 management in the critically ill emphasized the use of higher tidal and minute ventilation to avoid hypercapnia and its associated the risks. The demonstration that high lung stretch directly injures the lungs heralded the use of more protective ventilatory strategies that reduce lung stretch, and have been proven to improve survival in patients with ARDS. Consequently, hypercapnia – and its associated hypercapnic acidosis (HCA) - is prevalent in the critically ill, ‘permitted’ order to realize the benefits of lower lung stretch. Experimental and clinical investigations have generated key advances in our understanding of the effects of hypercapnia. Hypercapnia to be a potent biologic agent, with the potential to exert both beneficial and potentially harmful effects. Hypercapnia modulates the innate immune response, with inhibition of nuclear factor kappa-B, is a key transcriptional protein in injury, inflammation and repair, mediating diverse effects of hypercapnia. Advances in extracorporeal technologies have made possible the direct removal of CO2 while maintaining lung protective ventilation, a promising, though as yet unproven, approach. Consequently, it is important to understand the biology of hypercapnia, in order to best understand when hypercapnia should be encouraged, tolerated or avoided in patients with ARDS. 
Professor John Laffey, National University of Ireland, Galway, Ireland
Professor John Laffey, National University of Ireland, Galway, IrelandJohn Laffey is Professor of Anaesthesia and Intensive Care Medicine at the School of Medicine of the National University of Ireland, Galway and Galway University Hospitals. His research is focused on acute respiratory distress syndrome and sepsis, specifically on therapeutic potential of cell therapies for the critically ill. He also has a longstanding interest in the effects and mechanisms of action of carbon dioxide in the critically ill. |
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| 09:30 - 09:40 | Discussion |
| 09:40 - 10:10 |
CO2 sensing in the brain
The detection and regulated excretion of CO2, via breathing, is fundamental for homeostatic control of blood pH and preservation of life. Breathing is highly sensitive to the partial pressure of CO2 (PCO2) in blood. This vital physiological function was previously thought to depend exclusively upon the indirect changes in pH that follow the accumulation of CO2. However, evidence suggests that CO2 can have direct effects on breathing in addition to those of pH. Connexins are large-conductance hexameric plasma membrane channels. They can dock together to form a passageway between adjacent cells, a gap junction, to permit transfer of ions and small molecules. Connexin channels not docked to those in neighbouring cells form “hemichannels” and open to the extracellular space. Chemosensory cells at the surface of the medulla oblongata use hemichannels of connexin26 (Cx26) to detect CO2 and effect adaptive changes in breathing. Physiological levels CO2 cause hemichannels of Cx26 to open, permitting the release of the neurotransmitter ATP and excitation of the neural networks controlling breathing. CO2 most likely binds to Cx26 by carbamylating Lys125, which forms a salt bridge to Arg104 on a neighbouring subunit to open the hemichannel. Understanding the interaction of CO2 with Cx26 has enabled the rational design of a dominant negative subunit, dnCx26, which coassembles with wild type Cx26 to remove its CO2 sensitivity. Transduction with dnCx26 of the chemosensory cells in the medulla oblongata greatly reduces the chemosensitivity of breathing, thus directly linking the functional motif of CO2-binding to the physiological function of Cx26.
Professor Nicholas Dale, University of Warwick, UK
Professor Nicholas Dale, University of Warwick, UK |
| 10:10 - 10:20 | Discussion |
| 10:20 - 10:50 | Coffee |
| 10:50 - 11:20 |
CO2 diffusion inside leaves during photosynthesis
The rate of photosynthesis is very sensitive to the level of CO2 inside the chloroplasts, where photosynthesis takes place and CO2 is fixed by rubisco. CO2 first diffuses from the atmosphere through stomatal pores into the substomatal space. The CO2 must then cross internal airspace, cell walls, plasmalemma, cytoplasm, chloroplast envelope and part of the chloroplast stroma before it is fixed in the first step of the Calvin-Benson cycle. The combined conductance to CO2 transfer from substomatal cavities to the site of fixation is termed mesophyll conductance. In order to estimate mesophyll conductance, the exchange of CO2 from atmosphere into the leaf, together with the isotopic composition of the CO2 is measured. By exploiting Rubisco’s natural preference for 12CO2 over 13CO2, we can calculate mesophyll conductance from carbon isotopic discrimination. Mesophyll conductance is an important photosynthetic parameter that influences the amount of CO2 available for fixation and is a target for improving crop productivity. Understanding variations in mesophyll conductance across leaves, and what drives these changes, is essential for modelling how the manipulation of photosynthetic pathways may alter plant productivity. Using Nicotiana tabacum var. Samsun, we have investigated how mesophyll conductance, and other photosynthetic and leaf anatomy parameters, vary across leaf ages and throughout the canopy, with the goal of better informing plant productivity models. 
Dr Tory Clarke, Australian National University, Australia
Dr Tory Clarke, Australian National University, AustraliaDr Tory Clarke is a plant molecular biologist and physiologist at The Australian National University. She obtained her B.Biotech (Hons) at the University of Tasmania in 2008 and her PhD in Plant Molecular Biology at the University of Sydney in 2014. Tory has undertaken postdoctoral studies at Macquarie University and the Australian National University. Her current research focuses on understanding and improving the uptake of carbon dioxide into plant leaf cells to enhance photosynthetic efficiency and generate higher yielding crops. |
| 11:20 - 11:30 | Discussion |
| 11:30 - 12:00 |
Molecular and cellular mechanisms for CO2 sensing: lessons from aquatic organisms
My laboratory uses aquatic animals as models to study the evolution of acid-base sensing mechanisms at the molecular, cellular, and organismal levels. Because the internal fluids of aquatic organisms have lower CO2 and HCO3- levels and higher pH (“acid-base” parameters) compared to air-breathing vertebrates, their underlying acid-base sensing mechanisms must be tuned to different and specific set points. Kinetic assays on the evolutionary conserved acid-sensing enzyme soluble adenylyl cyclase (sAC) demonstrate a species-specific responsiveness to [HCO3-] that in each case matches physiologically relevant levels. For example, sAC’s HCO3- half-maximal stimulation is ~5 mM in sharks, ~10 mM in bony fishes and coral, which are lower than the ~20 mM reported in mammals. Additionally, aquatic animals routinely experience metabolic and environmental acid-base disturbances that can span >1 pH unit and >10-fold changes in [HCO3-]. Physiologically, this implies acid-base sensing plays essential and multiple homeostatic roles. Experimentally, this is advantageous because it allows imposing extreme (but physiologically relevant) acid-base challenges (i.e. 0-100 mM bicarbonate, pH 6.0-9.0) that maximize the magnitude of physiological responses and facilitate their detection. These approaches have led to the discovery of several novel physiological functions under sAC modulation in aquatic animals, including base secretion in shark gill epithelial cells, salt and fluid absorption across fish intestine, heart beat rate in hagfish, and pHi regulation in corals. In addition to their implications for organismal, environmental, and evolutionary physiology, these results provide clues about similar processes that might be under sAC control in humans and therefore might have biomedical relevance. 
Dr Martin Tresguerres, Scripps Institution of Oceanography, UC San Diego, USA
Dr Martin Tresguerres, Scripps Institution of Oceanography, UC San Diego, USADr Martin Tresguerres is an Associate Professor in Marine Biology at Scripps Institution of Oceanography, UC San Diego. His research studies molecular and cellular mechanisms for carbon dioxide, pH and bicarbonate sensing in diverse aquatic organisms, and their downstream homeostatic mechanisms in response to environmental and metabolic acid/base fluctuations. Some examples include coral calcification and metabolic communication between symbiotic partners, hagfish heart beat rate, shark blood acid/base regulation, and bony fish intestinal NaCl and fluid absorption. By studying a diverse array of organisms and physiological processes, Tresguerres’ work aims to identify evolutionary conserved acid/base sensing mechanisms. From an applied perspective, his work is relevant for determining organismal responses to environmental stress, for aquaculture, and as inspiration for biotechnological applications such as biodiesel production, carbon sequestration, and nanotechnology. Additionally, some acid/base sensing mechanisms are more easily studied in aquatic animals compared to mammals, and may inform biomedical applications. |
| 12:00 - 12:10 | Discussion |
Chair
Professor Martin Cann, Durham University, UK
Professor Martin Cann, Durham University, UK
Martin Cann did his PhD in Molecular Virology at the University of Reading and studied cyclic nucleotide signalling as a PDRA with Lonny Levin (Weill Medical College of Cornell University) and Dave Garbers (HHMI, University of Texas Southwestern). He was part of the team (with Lonny Levin) that identified the soluble adenylyl cyclase as a carbon-activated signalling molecule in mammals. In his own laboratory, at Durham University, he has had an interest in the general phenomenon of regulation of the cAMP signalling pathway by inorganic carbon. More recently he has been interested in carbon dioxide-mediated post-translational modifications and has established methodology to identify carbon dioxide-binding sites on protein.
| 13:20 - 13:50 |
Hypercapnia and suppression of lung host defense
Hypercapnia, or elevated PCO2 in blood and tissue, is an independent risk factor for mortality in patients with severe acute and advanced chronic lung disease. Bacterial and viral lung infections are often proximate events leading to poor clinical outcomes in such individuals. The Sporn group showed that exposure to elevated concentrations of CO2 inhibits innate immune gene expression and phagocytosis in macrophages, suggesting a causal role for hypercapnia in worse outcomes related to lung infection. Further, Sporn and colleagues showed that normoxic hypercapnia increased the mortality of bacterial pneumonia in mice. In addition, hypercapnia suppressed macrophage antiviral gene expression and increased the mortality of influenza A (IAV) infection in mice. Interestingly, hypercapnia also inhibited innate immune gene expression and increased the mortality of bacterial infection in Drosophila. A genome-wide RNAi screen then led to the identification of a transcription factor, zfh2, whose expression was required for CO2-induced immunosuppression in the fly in vitro and in vivo. The Sporn group generated a myeloid-specific mouse knockout of Zfhx3, the mammalian ortholog of zfh2, and finds that Zfhx3-deficient macrophages from the mutant are protected against hypercapnia-induced suppression of antiviral gene expression and increased growth of IAV in vitro. Further, the myeloid Zfhx3-deficient mouse is partially protected against IAV-induced lung injury. RNA-seq analysis of alveolar macrophages from IAV-infected mice reveals that host defense-related gene expression pathways downregulated by hypercapnia in the wild-type are upregulated in alveolar macrophages from Zfhx3-deficient mice. Future studies will further elucidate the molecular mechanism(s) by which hypercapnia suppresses lung host defense. 
Professor Peter Sporn, Northwestern University, USA
Professor Peter Sporn, Northwestern University, USADr Peter Sporn is Professor of Medicine and Cell and Developmental Biology at the Feinberg School of Medicine of Northwestern University, Chicago, Illinois, USA. He has longstanding clinical and research interests in inflammatory/immunologic lung disease and pulmonary infections. For the past decade, he has been studying the effects of elevated CO2 on the immune system, with a particular focus elucidate molecular mechanisms by which hypercapnia suppresses lung host defense against bacterial and viral pathogens. |
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| 13:50 - 14:00 | Discussion |
| 14:00 - 14:30 |
Carbon dioxide and hypercapnia in ventilation/perfusion regulation and inflammation
There has traditionally been a far greater emphasis in respiratory physiology and medicine on effects and roles of O2 than CO2 on the lung. This ‘oxycentric’ focus has relegated CO2 to that of the neglected step sister of the two gases, yet it has considerable influence on gas exchange efficiency and inflammation that are quite under-recognized. When either regional ventilation (VA) or perfusion (Q) varies, both O2 and CO2-dependent mechanisms are evoked to restore the balance of local blood flow and local gas flow. Matching of Q to changes in VA is accomplished by both hypoxic pulmonary vasoconstriction (HPV) and hypercapnic pulmonary vasoconstriction (HCPV), acting equally to limit perfusion into poorly ventilated areas. HCPV may be more important since it is active over the entire range of PCO2 in normal and diseased lungs, whereas as HPV is only engaged below a PO2 of 60 mmHg. In contrast to perfusion regulation, regulation of regional ventilation has no dependence on PO2. The changes in alveolar PCO2 caused by changes in Q act to alter VA by actions at the bronchial level by hypercapnic bronchodilation and at the parenchymal level by hypercapnic pneumo-relaxation causing changes in tissue compliance. These CO2-mediated effects are largely the result of the accompanying pH change which is accelerated by carbonic anhydrase in the lung. Beyond the benefits of CO2 on gas exchange efficiency and VA/Q matching, it also has anti-inflammatory effects that have the potential to mitigate lung injury from ischemia-reperfusion damage, acute respiratory distress syndrome and infections. 
Professor Erik Swenson, University of Washington, USA
Professor Erik Swenson, University of Washington, USADr Swenson is Professor of Medicine and Physiology at the University of Washington, Seattle, USA. He is the editor of High Altitude Medicine and Biology, section editor for Annals of the American Thoracic Society and serves on the editorial boards of Journal of Applied Physiology and American Journal of Respiratory and Critical Care Medicine. His clinical interests beyond pulmonary and critical care medicine are altitude, exercise and sports medicine and respiratory pharmacology. His research areas include 1) carbonic anhydrase and effects of its inhibitors in organ and cellular physiology, 2) human and animal adaptation and maladaptation to altitude and hypoxia, and 3) influences of carbon dioxide and acid-base disturbances on lung and other organ injuries and 4) comparative physiology of acid-base and hypoxia. He is a climber, cyclist, and swimmer, enjoys reading history, and volunteers to help disabled individuals to be outdoors in all seasons with adaptive equipment. |
| 14:30 - 14:40 | Discussion |
| 14:40 - 15:10 | Tea |
| 15:10 - 15:40 |
Stomatal CO2 detection
Applying either elevated atmospheric carbon dioxide concentrations or the drought hormone abscisic acid (ABA), to plant leaves brings about similar effects; they reduce the apertures of stomatal pores and reduce the number of stomata that form. These two stomatal responses allow plants to reduce their rate of transpiration and to conserve water. We therefore investigated whether the same signalling components might regulate stomatal responses to both [CO2] and ABA. Our results indicate that the stomatal [CO2] responses utilise components of the ABA signaling pathway including ABA biosynthesis and ABA receptors. Thus it appears possible that stomatal [CO2] responses are mediated by the ABA response pathway. However, we found no evidence for an increase in leaf ABA levels in response to elevated [CO2], suggesting that ABA either increases the sensitivity of the system to [CO2], or that any [CO2]-induced increase in ABA occurs specifically in the stomata. 
Professor Julie Gray, Sheffield University, UK
Professor Julie Gray, Sheffield University, UKJulie Gray carried out postdoctoral research into the molecular control of fruit ripening and flower pollination at Nottingham and Melbourne universities. Currently, she holds a personal chair in Plant Cell Signalling at the University of Sheffield where she studies how plants adapt to changing environmental conditions including drought and rising carbon dioxide levels. Her research focuses on the signalling pathways that regulate the development and closure of the leaf pores known as stomata which regulate plant water loss. Her group have identified and manipulated several stomatal components and recently demonstrated that plants with reduced stomatal density have enhanced drought tolerance. She has successfully used these findings from the model plant, Arabidopsis, to produce wheat and rice crop varieties with improved drought tolerance and water use efficiency. |
| 15:40 - 15:50 | Discussion |
| 15:50 - 16:20 |
CO2 and mammalian innate immunity
Carbon dioxide (CO2) is a product of aerobic metabolism that is mainly exhaled via the lungs to maintain stable paCO2 pressure (normocapnia) and blood pH. Hypercapnia (paCO2 >50mmHg) is a feature of chronic lung diseases, and permissive hypercapnia occurs in the intensive care setting as a feature of a protective ventilation strategy. Several in vitro and in vivo animal based studies have implicated CO2-dependent signaling in the suppression of immune and inflammatory signaling. Thus, these CO2-dependent signaling events may be detrimental in the context of a bacterial infection but of benefit in the context of uncontrolled inflammation. However, the molecular mechanisms underpinning CO2-dependent changes in gene expression are not well understood and are the focus of research in the Cummins lab. This presentation will initially outline investigations from the lab into how elevated CO2 modulates signaling, particularly within the NFkB pathway. Hypercapnia causes a marked change in the nuclear localisation and processing of key NFkB family members and modulates the expression of NFkB target genes. Dr Cummins will outline some of the recent RNA-seq and proteomic approaches taken by the group to understand the molecular mechanisms underpinning CO2-dependent regulation of inflammatory signaling. Finally, hypoxia and hypercapnia frequently co-exist given that O2 and CO2 are the substrate and product of aerobic metabolism respectively. Dr Cummins will describe important cross-talk between the O2 and CO2-sensing pathways and demonstrate that hypercapnia suppresses the cellular adaptive response to low oxygen in vitro and in vivo.
Dr Eoin Cummins, University College Dublin, Ireland
Dr Eoin Cummins, University College Dublin, IrelandEoin obtained a bachelor’s degree in Pharmacology (2002), and a PhD (2007) from University College Dublin (UCD). His early work focused the role of oxygen sensing hydroxylases in the context of intestinal inflammation. During his post-doctoral career Eoin became interested in the potential role for carbon dioxide as a modulator of inflammatory and immune signaling, in particular in relation to NFkB signaling. The first paper on this work was published in the Journal of Immunology in 2010. Eoin subsequently took up a short-term fellowship in Northwestern University, before returning to UCD and being appointed as an Assistant Professor in Physiology. He currently leads a Science Foundation Ireland funded research group focused on the effects of hypercapnia on inflammation and immunity. |
| 16:20 - 16:30 | Discussion |
| 16:30 - 17:00 | Closing discussion |