Image reconstruction in non-reciprocal broken-ray tomography.

Optical methods of biomedical tomographic imaging are of considerable interest due to their non-invasive nature and sensitivity to physiologically important markers. Similarly to other imaging modalities, optical methods can be enhanced by utilizing extrinsic contrast agents. Typically, these are fluorescent molecules, which can aggregate in regions of interest due to various mechanisms. In the current approaches to imaging, the intrinsic (related to the tissue) and extrinsic (related to the contrast agent) optical parameters are determined separately. This can result in errors, in particular, due to using simplified heuristic models for the spectral dependence of the optical parameters. Recently, we have developed the theory of non-reciprocal broken-ray tomography (NRBRT) for fluorescence imaging of weakly scattering systems. NRBRT enables simultaneous reconstruction of the fluorophore concentration as well as of the intrinsic optical attenuation coefficient at both the excitation and the emission wavelengths. Importantly, no assumption about the spectral dependence of the tissue optical properties is made in NRBRT. In this study, we perform numerical validation of NRBRT under realistic conditions using the Monte Carlo method to generate forward data. We demonstrate that NRBRT can be used for tomographic imaging of samples of up to four scattering lengths in size. The effects of physical characteristics of the detectors such as the area and the acceptance angle are also investigated.

Roles of RbcX in Carboxysome Biosynthesis in the Cyanobacterium Synechococcus elongatus PCC7942.

Rubisco is the essential enzyme mediating the fixation of atmospheric CO2 during photosynthesis. In cyanobacteria, Rubisco enzymes are densely packed and encapsulated in a specialized organelle known as the carboxysome. Well-defined Rubisco assembly and carboxysome formation are pivotal for efficient CO2 fixation. Numerous chaperone proteins, including RbcX, are essential for proper protein folding and Rubisco assembly. In this study, we investigated the in vivo function of RbcX in the cyanobacterium Synechococcus elongatus PCC 7942 (Syn7942) using molecular, biochemical, and live-cell fluorescence imaging approaches. Our results show that genetic deletion of the rbcX gene affects Rubisco abundance, as well as carboxysome formation and spatial distribution. Moreover, RbcX appears as one component of the carboxysome and shows a dynamic interaction with Rubisco enzymes. These in vivo observations provide insight into the role of RbcX from Syn7942 in mediating carboxysome assembly. Understanding the molecular mechanism underlying Rubisco assembly and carboxysome biogenesis will provide essential information required for engineering functional CO2-fixing complexes in heterogeneous organisms, especially plants, with the aim of boosting photosynthesis and agricultural productivity.

Regulation, sensory domains and roles of two Desulfovibrio desulfuricans ATCC27774 Crp family transcription factors, HcpR1 and HcpR2, in response to nitrosative stress.

In silico analyses identified a Crp/Fnr family transcription factor (HcpR) in sulfate-reducing bacteria that controls expression of the hcp gene, which encodes the hybrid cluster protein and contributes to nitrosative stress responses. There is only one hcpR gene in the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, but two copies in Desulfovibrio desulfuricans 27774, which can use nitrate as an alternative electron acceptor to sulfate. Structures of the D. desulfuricans hcpR1, hcpR2 and hcp operons are reported. We present evidence that hcp expression is regulated by HcpR2, not by HcpR1, and that these two regulators differ in both their DNA-binding site specificity and their sensory domains. HcpR1 is predicted to be a b-type cytochrome. HcpR1 binds upstream of the hcpR1 operon and its synthesis is regulated coordinately with hcp in response to NO. In contrast, hcpR2 expression was not induced by nitrate, nitrite or NO. HcpR2 is an iron-sulfur protein that reacts with NO and O2 . We propose that HcpR1 and HcpR2 use different sensory mechanisms to regulate subsets of genes required for defense against NO-induced nitrosative stress, and that diversification of signal perception and DNA recognition by these two proteins is a product of D. desulfuricans adaptation to its particular environmental niche.

Light Modulates the Biosynthesis and Organization of Cyanobacterial Carbon Fixation Machinery through Photosynthetic Electron Flow.

Cyanobacteria have evolved effective adaptive mechanisms to improve photosynthesis and CO2 fixation. The central CO2-fixing machinery is the carboxysome, which is composed of an icosahedral proteinaceous shell encapsulating the key carbon fixation enzyme, Rubisco, in the interior. Controlled biosynthesis and ordered organization of carboxysomes are vital to the CO2-fixing activity of cyanobacterial cells. However, little is known about how carboxysome biosynthesis and spatial positioning are physiologically regulated to adjust to dynamic changes in the environment. Here, we used fluorescence tagging and live-cell confocal fluorescence imaging to explore the biosynthesis and subcellular localization of ß-carboxysomes within a model cyanobacterium, Synechococcus elongatus PCC7942, in response to light variation. We demonstrated that ß-carboxysome biosynthesis is accelerated in response to increasing light intensity, thereby enhancing the carbon fixation activity of the cell. Inhibition of photosynthetic electron flow impairs the accumulation of carboxysomes, indicating a close coordination between ß-carboxysome biogenesis and photosynthetic electron transport. Likewise, the spatial organization of carboxysomes in the cell correlates with the redox state of photosynthetic electron transport chain. This study provides essential knowledge for us to modulate the ß-carboxysome biosynthesis and function in cyanobacteria. In translational terms, the knowledge is instrumental for design and synthetic engineering of functional carboxysomes into higher plants to improve photosynthesis performance and CO2 fixation.

Visualization of Bacterial Microcompartment Facet Assembly Using High-Speed Atomic Force Microscopy.

Bacterial microcompartments (BMCs) are proteinaceous organelles widespread among bacterial phyla. They compartmentalize enzymes within a selectively permeable shell and play important roles in CO2 fixation, pathogenesis, and microbial ecology. Here, we combine X-ray crystallography and high-speed atomic force microscopy to characterize, at molecular resolution, the structure and dynamics of BMC shell facet assembly. Our results show that preformed hexamers assemble into uniformly oriented shell layers, a single hexamer thick. We also observe the dynamic process of shell facet assembly. Shell hexamers can dissociate from and incorporate into assembled sheets, indicating a flexible intermolecular interaction. Furthermore, we demonstrate that the self-assembly and dynamics of shell proteins are governed by specific contacts at the interfaces of shell proteins. Our study provides novel insights into the formation, interactions, and dynamics of BMC shell facets, which are essential for the design and engineering of self-assembled biological nanoreactors and scaffolds based on BMC architectures.

T cell ageing: effects of age on development, survival & function.

Age associated decline of the immune system continues to be a major health concern. All components of innate and adaptive immunity are adversely affected to lesser or greater extent by ageing resulting in an overall decline of immunocompetence. As a result in the aged population, there is increased susceptibility to infection, poor responses to vaccination, and increased incidence of autoreactivity. There is an increasing focus on the role of T cells during ageing because of their impact on the overall immune responses. A steady decline in the production of fresh naïve T cells, more restricted T cell receptor (TCR) repertoire and weak activation of T cells are some of the effects of ageing. In this review we summarize our present understanding of the effects of ageing on naïve CD4 T cells and potential approaches for therapeutic interventions to restore protective immunity in the aged population.

Online Omics Platform Expedites Industrial Application of Halomonas bluephagenesis TD1.0.

Multi-omic data mining has the potential to revolutionize synthetic biology especially in non-model organisms that have not been extensively studied. However, tangible engineering direction from computational analysis remains elusive due to the interpretability of large datasets and the difficulty in analysis for non-experts. New omics data are generated faster than our ability to use and analyse results effectively, resulting in strain development that proceeds through classic methods of trial-and-error without insight into complex cell dynamics. Here we introduce a user-friendly, interactive website hosting multi-omics data. Importantly, this new platform allows non-experts to explore questions in an industrially important chassis whose cellular dynamics are still largely unknown. The web platform contains a complete KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis derived from principal components analysis, an interactive bio-cluster heatmap analysis of genes, and the Halomonas TD1.0 genome-scale metabolic (GEM) model. As a case study of the effectiveness of this platform, we applied unsupervised machine learning to determine key differences between Halomonas bluephagenesis TD1.0 cultivated under varied conditions. Specifically, cell motility and flagella apparatus are identified to drive energy expenditure usage at different osmolarities, and predictions were verified experimentally using microscopy and fluorescence labelled flagella staining. As more omics projects are completed, this landing page will facilitate exploration and targeted engineering efforts of the robust, industrial chassis H bluephagenesis for researchers without extensive bioinformatics background.

Chemoautotrophic production of gaseous hydrocarbons, bioplastics and osmolytes by a novel Halomonas species.

BACKGROUND: Production of relatively low value, bulk commodity chemicals and fuels by microbial species requires a step-change in approach to decrease the capital and operational costs associated with scaled fermentation. The utilisation of the robust and halophilic industrial host organisms of the genus Halomonas could dramatically decrease biomanufacturing costs owing to their ability to grow in seawater, using waste biogenic feedstocks, under non-sterile conditions. RESULTS: We describe the isolation of Halomonas rowanensis, a novel facultative chemoautotrophic species of Halomonas from a natural brine spring. We investigated the ability of this species to produce ectoine, a compound of considerable industrial interest, under heterotrophic conditions. Fixation of radiolabelled NaH14CO3 by H. rowanensis was confirmed in mineral medium supplied with thiosulfate as an energy source. Genome sequencing suggested carbon fixation proceeds via a reductive tricarboxylic acid cycle, and not the Calvin-Bensen-Bassham cycle. The mechanism of energy generation to support chemoautotrophy is unknown owing to the absence of an annotated SOX-based thiosulfate-mediated energy conversion system. We investigated further the biotechnological potential of the isolated H. rowanensis by demonstrating production of the gaseous hydrocarbon (bio-propane), bioplastics (poly-3-hydroxybutyrate) and osmolytes (ectoine) under heterotrophic and autotrophic CO2 fixation growth conditions. CONCLUSIONS: This proof-of-concept study illustrates the value of recruiting environmental isolates as industrial hosts for chemicals biomanufacturing, where CO2 utilisation could replace, or augment, the use of biogenic feedstocks in non-sterile, industrialised bioreactors.

Combinatorial use of environmental stresses and genetic engineering to increase ethanol titres in cyanobacteria.

Current industrial bioethanol production by yeast through fermentation generates carbon dioxide. Carbon neutral bioethanol production by cyanobacteria uses biological fixation (photosynthesis) of carbon dioxide or other waste inorganic carbon sources, whilst being sustainable and renewable. The first ethanologenic cyanobacterial process was developed over two decades ago using Synechococcus elongatus PCC 7942, by incorporating the recombinant pdc and adh genes from Zymomonas mobilis. Further engineering has increased bioethanol titres 24-fold, yet current levels are far below what is required for industrial application. At the heart of the problem is that the rate of carbon fixation cannot be drastically accelerated and carbon partitioning towards bioethanol production impacts on cell fitness. Key progress has been achieved by increasing the precursor pyruvate levels intracellularly, upregulating synthetic genes and knocking out pathways competing for pyruvate. Studies have shown that cyanobacteria accumulate high proportions of carbon reserves that are mobilised under specific environmental stresses or through pathway engineering to increase ethanol production. When used in conjunction with specific genetic knockouts, they supply significantly more carbon for ethanol production. This review will discuss the progress in generating ethanologenic cyanobacteria through chassis engineering, and exploring the impact of environmental stresses on increasing carbon flux towards ethanol production.

Naive CD4 T cells from aged mice show enhanced death upon primary activation.

Poor T cell immunity is one of the many defects seen in elderly humans and aged (Ad) mice. We report that naive CD4 T cells from aged mice (ANCD4 cells) showed greater apoptosis upon primary activation than those from young (Yg) mice, with loss of mitochondrial membrane potential, poor activation of Rel family transcription factors and increased DNA damage. Their ability to enhance glycolysis, produce lactate and induce autophagy following activation was also compromised. ANCD4 cells remained susceptible to death beyond first cell division. Activated ANCD4 cells also showed poor transition to a 'central memory' (CM) CD44(high), CD62L(high) phenotype in vitro. This correlated with low proportions of CM cells in Ad mice in vivo. Functionally, too, IFN-gamma responses recalled from T cells of immunized Ad mice, poor to begin with, worsened with time as compared with Yg mice. Thus, ANCD4 cells handle activation-associated stress very poorly due to multiple defects, possibly contributing to poor formation of long-lasting memory.

Molecular simulations unravel the molecular principles that mediate selective permeability of carboxysome shell protein.

Bacterial microcompartments (BMCs) are nanoscale proteinaceous organelles that encapsulate enzymes from the cytoplasm using an icosahedral protein shell that resembles viral capsids. Of particular interest are the carboxysomes (CBs), which sequester the CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to enhance carbon assimilation. The carboxysome shell serves as a semi-permeable barrier for passage of metabolites in and out of the carboxysome to enhance CO2 fixation. How the protein shell directs influx and efflux of molecules in an effective manner has remained elusive. Here we use molecular dynamics and umbrella sampling calculations to determine the free-energy profiles of the metabolic substrates, bicarbonate, CO2 and ribulose bisphosphate and the product 3-phosphoglycerate associated with their transition through the major carboxysome shell protein CcmK2. We elucidate the electrostatic charge-based permeability and key amino acid residues of CcmK2 functioning in mediating molecular transit through the central pore. Conformational changes of the loops forming the central pore may also be required for transit of specific metabolites. The importance of these in-silico findings is validated experimentally by site-directed mutagenesis of the key CcmK2 residue Serine 39. This study provides insight into the mechanism that mediates molecular transport through the shells of carboxysomes, applicable to other BMCs. It also offers a predictive approach to investigate and manipulate the shell permeability, with the intent of engineering BMC-based metabolic modules for new functions in synthetic biology.

Renewable and tuneable bio-LPG blends derived from amino acids.

BACKGROUND: Microbial biorefinery approaches are beginning to define renewable and sustainable routes to clean-burning and non-fossil fuel-derived gaseous alkanes (known as 'bio-LPG'). The most promising strategies have used a terminal fatty acid photodecarboxylase, enabling light-driven propane production from externally fed waste butyric acid. Use of Halomonas (a robust extremophile microbial chassis) with these pathways has enabled bio-LPG production under non-sterile conditions and using waste biomass as the carbon source. Here, we describe new engineering approaches to produce next-generation pathways that use amino acids as fuel precursors for bio-LPG production (propane, butane and isobutane blends). RESULTS: Multiple pathways from the amino acids valine, leucine and isoleucine were designed in E. coli for the production of propane, isobutane and butane, respectively. A branched-chain keto acid decarboxylase-dependent pathway utilising fatty acid photodecarboxylase was the most effective route, generating higher alkane gas titres over alternative routes requiring coenzyme A and/or aldehyde deformylating oxygenase. Isobutane was the major gas produced in standard (mixed amino acid) medium, however valine supplementation led to primarily propane production. Transitioning pathways into Halomonas strain TQ10 enabled fermentative production of mixed alkane gases under non-sterile conditions on simple carbon sources. Chromosomal integration of inducible (~ 180 mg/g cells/day) and constitutive (~ 30 mg/g cells/day) pathways into Halomonas generated production strains shown to be stable for up to 7 days. CONCLUSIONS: This study highlights new microbial pathways for the production of clean-burning bio-LPG fuels from amino acids. The use of stable Halomonas production strains could lead to gas production in the field under non-sterile conditions following process optimisation.

Promoter engineering for microbial bio-alkane gas production.

Successful industrial biotechnological solutions to biofuels and other chemicals production rely on effective competition with existing lower-cost natural sources and synthetic chemistry approaches enabled by adopting low-cost bioreactors and processes. This is achievable by mobilizing Halomonas as a next generation industrial chassis, which can be cultivated under non-sterile conditions. To increase the cost effectiveness of an existing sustainable low carbon bio-propane production strategy, we designed and screened a constitutive promoter library based on the known strong porin promoter from Halomonas. Comparative studies were performed between Escherichia coli and Halomonas using the reporter gene red fluorescent protein (RFP). Later studies with a fatty acid photodecarboxylase-RFP fusion protein demonstrated tuneable propane production in Halomonas and E. coli, with an ∼8-fold improvement in yield over comparable isopropyl-ß-D-thiogalactoside-inducible systems. This novel set of promoters is a useful addition to the synthetic biology toolbox for future engineering of Halomonas to make chemicals and fuels.

Self-Assembly Stability and Variability of Bacterial Microcompartment Shell Proteins in Response to the Environmental Change.

Bacterial microcompartments (BMCs) are proteinaceous self-assembling organelles that are widespread among the prokaryotic kingdom. By segmenting key metabolic enzymes and pathways using a polyhedral shell, BMCs play essential roles in carbon assimilation, pathogenesis, and microbial ecology. The BMC shell is composed of multiple protein homologs that self-assemble to form the defined architecture. There is tremendous interest in engineering BMCs to develop new nanobioreactors and molecular scaffolds. Here, we report the quantitative characterization of the formation and self-assembly dynamics of BMC shell proteins under varying pH and salt conditions using high-speed atomic force microscopy (HS-AFM). We show that 400-mM salt concentration is prone to result in larger single-layered shell patches formed by shell hexamers, and a higher dynamic rate of hexamer self-assembly was observed at neutral pH. We also visualize the variability of shell proteins from hexameric assemblies to fiber-like arrays. This study advances our knowledge about the stability and variability of BMC protein self-assemblies in response to microenvironmental changes, which will inform rational design and construction of synthetic BMC structures with the capacity of remodeling their self-assembly and structural robustness. It also offers a powerful toolbox for quantitatively assessing the self-assembly and formation of BMC-based nanostructures in biotechnology applications.

Nanoscale Visualization of Bacterial Microcompartments Using Atomic Force Microscopy.

Bacterial microcompartments (BMCs) are polyhedral protein organelles in many prokaryotes, playing significant roles in metabolic enhancement. Due to their self-assembly and modularity nature, BMCs have gained increased interest in recent years, with the intent of constructing new nanobioreactors and scaffolding to promote cellular metabolisms and molecule delivery. In this chapter, we describe the technique of atomic force microscopy (AFM) as a method to study the self-assembly dynamics and physical properties of BMCs. We focus on the sample preparation, the measurement procedure, and the data analysis for high-speed AFM imaging and nanoindentation-based spectroscopy, which were used to determine the assembly dynamics of BMC shell proteins and the nanomechanics of intact BMC structures, respectively. The described methods could be applied to the study of other types of self-assembling biological organelles.

Engineering and Modulating Functional Cyanobacterial CO2-Fixing Organelles.

Bacterial microcompartments (BMCs) are proteinaceous organelles widespread among bacterial phyla and provide a means for compartmentalizing specific metabolic pathways. They sequester catalytic enzymes from the cytoplasm, using an icosahedral proteinaceous shell with selective permeability to metabolic molecules and substrates, to enhance metabolic efficiency. Carboxysomes were the first BMCs discovered and their unprecedented capacity of CO2 fixation allows cyanobacteria to make a significant contribution to global carbon fixation. There is an increasing interest in utilizing synthetic biology to construct synthetic carboxysomes in new hosts, i.e., higher plants, to enhance carbon fixation and productivity. Here, we report the construction of a synthetic operon of the ß-carboxysome from the cyanobacterium Synechococcus elongatus PCC7942 to generate functional ß-carboxysome-like structures in Escherichia coli. The protein expression, structure, assembly, and activity of synthetic ß-carboxysomes were characterized in depth using confocal, electron and atomic force microscopy, proteomics, immunoblot analysis, and enzymatic assays. Furthermore, we examined the in vivo interchangeability of ß-carboxysome building blocks with other BMC components. To our knowledge, this is the first production of functional ß-carboxysome-like structures in heterologous organisms. It provides important information for the engineering of fully functional carboxysomes and CO2-fixing modules in higher plants. The study strengthens our synthetic biology toolbox for generating BMC-based organelles with tunable activities and new scaffolding biomaterials for metabolic improvement and molecule delivery.

Coordinated response of the Desulfovibrio desulfuricans 27774 transcriptome to nitrate, nitrite and nitric oxide.

The sulfate reducing bacterium Desulfovibrio desulfuricans inhabits both the human gut and external environments. It can reduce nitrate and nitrite as alternative electron acceptors to sulfate to support growth. Like other sulphate reducing bacteria, it can also protect itself against nitrosative stress caused by NO generated when nitrite accumulates. By combining in vitro experiments with bioinformatic and RNA-seq data, metabolic responses to nitrate or NO and how nitrate and nitrite reduction are coordinated with the response to nitrosative stress were revealed. Although nitrate and nitrite reduction are tightly regulated in response to substrate availability, the global responses to nitrate or NO were largely regulated independently. Multiple NADH dehydrogenases, transcription factors of unknown function and genes for iron uptake were differentially expressed in response to electron acceptor availability or nitrosative stress. Amongst many fascinating problems for future research, the data revealed a YtfE orthologue, Ddes_1165, that is implicated in the repair of nitrosative damage. The combined data suggest that three transcription factors coordinate this regulation in which NrfS-NrfR coordinates nitrate and nitrite reduction to minimize toxicity due to nitrite accumulation, HcpR1 serves a global role in regulating the response to nitrate, and HcpR2 regulates the response to nitrosative stress.

Direct characterization of the native structure and mechanics of cyanobacterial carboxysomes.

Carboxysomes are proteinaceous organelles that play essential roles in enhancing carbon fixation in cyanobacteria and some proteobacteria. These self-assembling organelles encapsulate Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase using a protein shell structurally resembling an icosahedral viral capsid. The protein shell serves as a physical barrier to protect enzymes from the cytosol and a selectively permeable membrane to mediate transport of enzyme substrates and products. The structural and mechanical nature of native carboxysomes remain unclear. Here, we isolate functional ß-carboxysomes from the cyanobacterium Synechococcus elongatus PCC7942 and perform the first characterization of the macromolecular architecture and inherent physical mechanics of single ß-carboxysomes using electron microscopy, atomic force microscopy (AFM) and proteomics. Our results illustrate that the intact ß-carboxysome comprises three structural domains, a single-layered icosahedral shell, an inner layer and paracrystalline arrays of interior Rubisco. We also observe the protein organization of the shell and partial ß-carboxysomes that likely serve as the ß-carboxysome assembly intermediates. Furthermore, the topography and intrinsic mechanics of functional ß-carboxysomes are determined in native conditions using AFM and AFM-based nanoindentation, revealing the flexible organization and soft mechanical properties of ß-carboxysomes compared to rigid viruses. Our study provides new insights into the natural characteristics of ß-carboxysome organization and nanomechanics, which can be extended to diverse bacterial microcompartments and are important considerations for the design and engineering of functional carboxysomes in other organisms to supercharge photosynthesis. It offers an approach for inspecting the structural and mechanical features of synthetic metabolic organelles and protein scaffolds in bioengineering.

Revised Genome Sequence of the Purple Photosynthetic Bacterium Blastochloris viridis.

Blastochloris viridis is a unique anaerobic, phototrophic purple bacterium that produces bacteriochlorophyll b. Here we report an improved genome sequence of Blastochloris viridis DSM133, which is instrumental to the studies of photosynthesis, metabolic versatility, and genetic engineering of this microorganism.

Fluid prescription practices of anesthesiologists managing patients undergoing elective colonoscopy: an observational study.

BACKGROUND: Routine fluid prescription is common practice amongst anesthesiologists caring for patients undergoing colonoscopy. However there is limited information about routine procedural fluid prescription practices of anesthesiologists in this setting. Routine fluid administration may also have important pharmaco-economic implications for the health care budget. Therefore we performed a prospective observational study assessing the fluid prescription practices of anesthesiologists caring for patients undergoing elective colonoscopy. METHODS: With Institutional Review Board approval, adult patients receiving procedural fluid intervention during elective colonoscopy were included. DATA COLLECTED: size of intravenous cannula inserted, volumes of fluid administered, adverse events, procedure duration, and pharmaco-economic costs associated with fluid prescription. Anesthesiologists and gastroenterologists were blinded to the study. RESULTS: We collected data on 289 patients who received fluid prescription by their attending anesthesiologist. Median patient age: 48 yrs (range 18-83), gender: 174 (60%) female; median duration of procedure: 24 minutes (range 12-48). Cannula size: 181 (63%) patients received a 22G cannula or smaller. Median volume of fluid administered during the colonoscopy was 325 ml (range 0 to 1000 ml). Median duration of the procedure: 25 minutes (range 12 to 48 minutes). Median volume of fluid administered in the post anaesthesia recovery unit: 450 ml (range 0 to 1000 ml). Fifteen patients (5%) became hypotensive during the procedure and two patients (<1%) developed hypotension in the PACU. There was no difference in the median fluid requirements between patients with hypotension and those without. Fluid volumes were strongly associated with increasing cannula diameter (p = 0.0001), however there was no association between fluid volumes administered and vasopressor use, peri-procedural adverse events, or procedure duration. At our institution fluid therapy currently cost about AUD$4.90 per patient: 1 L crystalloid $1.18 and fluid delivery set $3.77 Our institution performs over 9000 endoscopic procedures annually with fluid therapy costing about $45,000/year. CONCLUSIONS: Routine fluid prescription by anesthesiologists managing patients undergoing colonoscopy was ineffective with low actual fluid volumes delivered during the procedure. There was no association between volumes of fluid delivered and procedural hypotension, adverse events, or procedure duration. Anesthesiologists should question the clinical and pharmaco-economic value of routine fluid administration for patients undergoing elective endoscopy.