Susceptibility of the Different Oxygen-Sensing Probes to Interferences in Respirometric Bacterial Assays with Complex Media.

Respirometric microbial assays are gaining popularity, but their uptake is limited by the availability of optimal O2 sensing materials and the challenge of validating assays with complex real samples. We conducted a comparative evaluation of four different O2-sensing probes based on Pt-porphyrin phosphors in respirometric bacterial assays performed on standard time-resolved fluorescence reader. The macromolecular MitoXpress, nanoparticle NanO2 and small molecule PtGlc4 and PtPEG4 probes were assessed with E. coli cells in five growth media: nutrient broth (NB), McConkey (MC), Rapid Coliform ChromoSelect (RCC), M-Lauryl lauryl sulfate (MLS), and Minerals-Modified Glutamate (MMG) media. Respiration profiles of the cells were recorded and analyzed, along with densitometry profiles and quenching studies of individual media components. This revealed several limiting factors and interferences impacting assay performance, which include probe quenched lifetime, instrument temporal resolution, inner filter effects (mainly by indicator dyes), probe binding to lipophilic components, and dynamic and static quenching by media components. The study allowed for the ranking of the probes based on their ruggedness, resilience to interferences and overall performance in respirometric bacterial assays. The 'shielded' probe NanO2 outperformed the established MitoXpress probe and the small molecule probes PtGlc4 and PtPEG4.

Analysis of tumour oxygenation in model animals on a phosphorescence lifetime based macro-imager.

Monitoring of tissue O2 is essential for cancer development and treatment, as hypoxic tumour regions develop resistance to radio- and chemotherapy. We describe a minimally invasive technique for the monitoring of tissue oxygenation in developing grafted tumours, which uses the new phosphorescence lifetime based Tpx3Cam imager. CT26 cells stained with a near-infrared emitting nanoparticulate O2 probe NanO2-IR were injected into mice to produce grafted tumours with characteristic phosphorescence. The tumours were allowed to develop for 3, 7, 10 and 17 days, with O2 imaging experiments performed on live and euthanised animals at different time points. Despite a marked trend towards decreased O2 in dead animals, their tumour areas produced phosphorescence lifetime values between 44 and 47 µs, which corresponded to hypoxic tissue with 5-20 µM O2. After the O2 imaging in animals, confocal Phosphorescence Lifetime Imaging Microscopy was conducted to examine the distribution of NanO2-IR probe in the tumours, which were excised, fixed and sliced for the purpose. The probe remained visible as bright and discrete 'islands' embedded in the tumour tissue until day 17 of tumour growth. Overall, this O2 macro-imaging method using NanO2-IR holds promise for long-term studies with grafted tumours in live animal models, providing quantitative 2D mapping of tissue O2.

Combining the oxygen sensor based respirometry and 16S rRNA amplicon sequencing for the analysis of microbiota in commercial mince products.

In this study, rapid respirometric microbial testing was combined with 16S rRNA amplicon sequencing, to assess the composition of microbiota in a total of 64 samples of commercial beef, turkey, lamb and pork mince. The O2 sensor-based respirometry system, while producing the anticipated total aerobic viable counts (TVC) data and patterns for most samples, also revealed unusual (linear) respiration profiles for some samples, mostly lamb and pork mince. The TVC values for beef mince, produced by respirometry and calculated using the available calibration equation, correlated well with the conventional plate counting method, ISO 4833-1:2013, 2013, while for the other species the correlation was less good. These effects, not observed in previous studies employing various food matrices, require further investigation. Using the same samples (crude homogenates) as in respirometry, the whole microbiome was also analysed by 16S rRNA amplicon sequencing for each mince-type. The sequencing showed an overall decrease in alpha diversity over shelf-life, with lamb and pork mince maintaining a proportion of rare taxa. Some taxa exhibited significant changes in abundance over shelf-life and after the respirometric analysis, with beef mince exhibiting a decrease in aerobic bacteria and an increase in facultative anaerobes. Beta diversity was also seen to depend on mince-type. Thus, the combined use of respirometry and sequencing techniques shows promise as a useful and unique analytical approach for food quality and safety evaluation, However, more data points and in-depth analysis are required to back up the findings of this initial study.

Oxygen Sensor-Based Respirometry and the Landscape of Microbial Testing Methods as Applicable to Food and Beverage Matrices.

The current status of microbiological testing methods for the determination of viable bacteria in complex sample matrices, such as food samples, is the focus of this review. Established methods for the enumeration of microorganisms, particularly, the 'gold standard' agar plating method for the determination of total aerobic viable counts (TVC), bioluminescent detection of total ATP, selective molecular methods (immunoassays, DNA/RNA amplification, sequencing) and instrumental methods (flow cytometry, Raman spectroscopy, mass spectrometry, calorimetry), are analyzed and compared with emerging oxygen sensor-based respirometry techniques. The basic principles of optical O2 sensing and respirometry and the primary materials, detection modes and assay formats employed are described. The existing platforms for bacterial cell respirometry are then described, and examples of particular assays are provided, including the use of rapid TVC tests of food samples and swabs, the toxicological screening and profiling of cells and antimicrobial sterility testing. Overall, O2 sensor-based respirometry and TVC assays have high application potential in the food industry and related areas. They detect viable bacteria via their growth and respiration; the assay is fast (time to result is 2-8 h and dependent on TVC load), operates with complex samples (crude homogenates of food samples) in a simple mix-and-measure format, has low set-up and instrumentation costs and is inexpensive and portable.

Fluorescence Lifetime Macro Imager for Biomedical Applications.

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ranging from solid-state, O2-sensitive coatings to live animal tissue samples stained with soluble O2-sensitive probes. In particular, the nanoparticle-based near-infrared probe NanO2-IR, which is excitable with a 625 nm light-emitting diode (LED) and emits at 760 nm, was used. The imaging system is based on the Timepix3 camera (Tpx3Cam) and the opto-mechanical adaptor, which also houses an image intensifier. O2 phosphorescence lifetime imaging microscopy (PLIM) is commonly required for various studies, but current platforms have limitations in their accuracy, general flexibility, and usability. The system presented here is a fast and highly sensitive imager, which is built on an integrated optical sensor and readout chip module, Tpx3Cam. It is shown to produce high-intensity phosphorescence signals and stable lifetime values from surface-stained intestinal tissue samples or intraluminally stained fragments of the large intestine and allows the detailed mapping of tissue O2 levels in about 20 s or less. Initial experiments on the imaging of hypoxia in grafted tumors in unconscious animals are also presented. We also describe how the imager can be re-configured for use with O2-sensitive materials based on Pt-porphyrin dyes using a 390 nm LED for the excitation and a bandpass 650 nm filter for emission. Overall, the PLIM imager was found to produce accurate quantitative measurements of lifetime values for the probes used and respective two-dimensional maps of the O2 concentration. It is also useful for the metabolic imaging of ex vivo tissue models and live animals.

Heterosubstituted Derivatives of PtPFPP for O2 Sensing and Cell Analysis: Structure-Activity Relationships.

Biological applications of phosphorescent probes for sensing molecular oxygen (O2) and bioimaging have gained popularity, but their choice is rather limited. We describe a family of new heterosubstituted phosphorescent bioprobes based on the Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye. The probes are produced by simple click modification of its para-fluorine atoms with thiols, such as 1/2-thio-glucose, thio-poly(ethylene glycol) (PEG), or cysteamine. The probes were designed to have one cell-targeting moiety and three polar moieties forming a hydrophilic shell. Their chemical synthesis and purification were optimized to produce high reaction yields and easy scale-up. The ability to perform as cell-permeable or -impermeable probes was tuned by the polarity and molecular charge of the bioconjugate. The new PtPFPP derivatives were characterized for their spectral properties and cell-penetrating ability in the experiments with mammalian cell cultures, using a time-resolved fluorescence reader and PLIM imaging detection. Structure-activity relationships were established. Thus, the tri- and tetra-PEGylated structures showed low cell internalization allowing their use as extracellular probes, while cysteamine derivatives performed as efficient intracellular probes. No significant cytotoxicity was observed for all of the probes under the experimental conditions used.

Ghrelin rapidly elevates protein synthesis in vitro by employing the rpS6K-eEF2K-eEF2 signalling axis.

Activated ghrelin receptor GHS-R1α triggers cell signalling pathways that modulate energy homeostasis and biosynthetic processes. However, the effects of ghrelin on mRNA translation are unknown. Using various reporter assays, here we demonstrate a rapid elevation of protein synthesis in cells within 15-30 min upon stimulation of GHS-R1α by ghrelin. We further show that ghrelin-induced activation of translation is mediated, at least in part, through the de-phosphorylation (de-suppression) of elongation factor 2 (eEF2). The levels of eEF2 phosphorylation at Thr56 decrease due to the reduced activity of eEF2 kinase, which is inhibited via Ser366 phosphorylation by rpS6 kinases. Being stress-susceptible, the ghrelin-mediated decrease in eEF2 phosphorylation can be abolished by glucose deprivation and mitochondrial uncoupling. We believe that the observed burst of translation benefits rapid restocking of neuropeptides, which are released upon GHS-R1α activation, and represents the most time- and energy-efficient way of prompt recharging the orexigenic neuronal circuitry.

Mitochondrial complex IV defects induce metabolic and signaling perturbations that expose potential vulnerabilities in HCT116 cells.

Mutations in genes encoding cytochrome c oxidase (mitochondrial complex IV) subunits and assembly factors [e.g., synthesis of cytochrome c oxidase 2 (SCO2)] are linked to severe metabolic syndromes. Notwithstanding that SCO2 is under transcriptional control of tumor suppressor p53, the role of mitochondrial complex IV dysfunction in cancer metabolism remains obscure. Herein, we demonstrate that the loss of SCO2 in HCT116 colorectal cancer cells leads to significant metabolic and signaling perturbations. Specifically, abrogation of SCO2 increased NAD+ regenerating reactions and decreased glucose oxidation through citric acid cycle while enhancing pyruvate carboxylation. This was accompanied by a reduction in amino acid levels and the accumulation of lipid droplets. In addition, SCO2 loss resulted in hyperactivation of the insulin-like growth factor 1 receptor (IGF1R)/AKT axis with paradoxical downregulation of mTOR signaling, which was accompanied by increased AMP-activated kinase activity. Accordingly, abrogation of SCO2 expression appears to increase the sensitivity of cells to IGF1R and AKT, but not mTOR inhibitors. Finally, the loss of SCO2 was associated with reduced proliferation and enhanced migration of HCT116 cells. Collectively, herein we describe potential adaptive signaling and metabolic perturbations triggered by mitochondrial complex IV dysfunction.

A sensor-based system for rapid on-site testing of microbial contamination in meat samples and carcasses.

AIMS: To develop an oxygen sensor-based method for testing total aerobic viable counts (TVC) in raw meat samples and cattle carcass swabs, which is rapid, simple, affordable, provides good sensitivity and analytical performance and allows on-site use. METHODS AND RESULTS: The test uses the same sample preparation procedure as the established plate counting TVC method for meat samples and carcasses, ISO4833-1:2013. After this liquid samples are transferred into standard 25-ml vials with built-in phosphorescent O2  sensors and incubated on a block heater with hourly readings of sensor signals with a handheld reader, to determine signal threshold time (TT, hours) for each sample. The method is demonstrated with the quantification of TVC in industrial cuts of raw beef meat (CFU per g) and carcass swabs (CFU per cm2 ). Calibration curves were generated, which give the following analytical equations for calculating the TVC load in unknown samples from measured TT values: TVC [Log(CFU per cm2 )] = 7.83-0.73*TT(h) and TVC [Log(CFU per g)] = 8.74-0.70*TT(h) for the carcass swabs and meat samples respectively. The new tests show good correlation with the ISO methods, with correlation coefficients 0.85 and 0.83 respectively. The testing requires no dilutions, covers the ranges 2-7 Log(CFU per g) for the meat samples and 1-7 Log(CFU per cm2 ) for carcass swabs, and has time to result 1-10 h with faster detection of more contaminated samples. CONCLUSIONS: The sensor-based testing demonstrates simplicity, high speed, sample throughput and automation. It can provide a straightforward replacement for the conventional TVC tests, which are time consuming, laborious and have time to result of 48-72 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The method(s) can be adopted by the meat industry and research labs, and used to improve microbial quality and safety of meat products and processes.

A Simple Sensor System for Onsite Monitoring of O2 in Vacuum-Packed Meats during the Shelf Life.

Vacuum packaging (VP) is used to reduce exposure of retail meat samples to ambient oxygen (O2) and preserve their quality. A simple sensor system produced from commercial components is described, which allows for non-destructive monitoring of the O2 concentration in VP raw meat samples. Disposable O2 sensor inserts were produced by spotting small aliquots of the cocktail of the Pt-benzoporphyrin dye and polystyrene in ethyl acetate onto pieces of a PVDF membrane and allowing them to air-dry. These sensor dots were placed on top of the beef cuts and vacuum-packed. A handheld reader, FirestinGO2, was used to read nondestructively the sensor phase shift signals (dphi°) and relate them to the O2 levels in packs (kPa or %). The system was validated under industrial settings at a meat processing plant to monitor O2 in VP meat over nine weeks of shelf life storage. The dphi° readings from individual batch-calibrated sensors were converted into the O2 concentration by applying the following calibration equation: O2 (%) = 0.034 * dphi°2 - 3.413 * dphi° + 85.02. In the VP meat samples, the O2 levels were seen to range between 0.12% and 0.27%, with the sensor dphi signals ranging from 44.03° to 56.02°. The DIY sensor system demonstrated ease of use on-site, fast measurement time, high sample throughput, low cost and flexibility.

Cell Energy Budget Platform for Multiparametric Assessment of Cell and Tissue Metabolism.

Specific bioenergetic signature reports on the current metabolic state of the cell, which may be affected by metabolic rearrangement, dysfunction or dysregulation of relevant signaling pathways, altered physiological condition or energy stress. A combined analysis of respiration , glycolytic flux, Krebs cycle activity, ATP levels, and total biomass allows informative initial assessment. Such simple, high-throughput, multiparametric methodology, called cell energy budget (CEB ) platform, is presented here and demonstrated with particular cell and tissue models. The CEB uses a commercial fluorescent lanthanide probe pH-Xtra™ to measure extracellular acidification (ECA) associated with lactate (L-ECA) and combined lactate/CO2 (T-ECA), a phosphorescent probe MitoXpress®-Xtra to measure oxygen consumption rate (OCR), a bioluminescent ATP kit, and an absorbance-based total protein assay. All the assays are performed on a standard multi-label reader. Using the same readouts, the CEB approach can be extended to more detailed mechanistic studies, by targeting specific pathways in cell bioenergetics and measuring other cellular parameters, such as NAD(P)H, Ca2+, mitochondrial pH, membrane potential, redox state, with conventional fluorescent or luminescent probes.

Facile biosensor-based system for on-site quantification of total viable counts in food and environmental swabs.

We describe a new biosensor platform for rapid and simple quantification of total aerobic viable counts of bacteria (TVC) in food and environmental swabs by oxygen respirometry. The system uses disposable swab vials with phosphorescent oxygen sensors integrated in the bottom part, a small block heater/incubator and a handheld sensor reader. In the testing, groups of 1-20 swabs samples were prepared using the standard method (ISO, 18593:2018) in sensor vials, which were then incubated at 30 °C and measured hourly in a contactless, non-invasive manner. The measurements reveal time profiles of dissolved O2 in each sample vial, from which Threshold Time of sensor signal was determined and then TVC values (CFU/cm2) were calculated using the calibration equation. The method covers the range of 0.65-7.87 Log (CFU/cm2) and produces results in 1-8 hrs. The test was validated with swab samples from surfaces contaminated with E. coli, with whole meat microbiota, and with real environmental swabs. The results showed no statistically significant difference with the reference method which takes 48-72 h. The swab testing platform is fast and accurate, simple (sample-and-measure), portable, low cost (<$5k), requires no serial dilutions and is suitable for on-site deployment and use.

Mapping O2 concentration in ex-vivo tissue samples on a fast PLIM macro-imager.

O2 PLIM microscopy was employed in various studies, however current platforms have limitations in sensitivity, image acquisition speed, accuracy and general usability. We describe a new PLIM imager based on the Timepix3 camera (Tpx3cam) and its application for imaging of O2 concentration in various tissue samples stained with a nanoparticle based probe, NanO2-IR. Upon passive staining of mouse brain, lung or intestinal tissue surface with minute quantities of NanO2-IR or by microinjecting the probe into the lumen of small or large intestine fragments, robust phosphorescence intensity and lifetime signals were produced, which allow mapping of O2 in the tissue within 20 s. Inhibition of tissue respiration or limitation of O2 diffusion to tissue produced the anticipated increases or decreases in O2 levels, respectively. The difference in O2 concentration between the colonic lumen and air-exposed serosal surface was around 140 µM. Furthermore, subcutaneous injection of 5 µg of the probe in intact organs (a paw or tail of sacrificed mice) enabled efficient O2 imaging at tissue depths of up to 0.5 mm. Overall, the PLIM imager holds promise for metabolic imaging studies with various ex vivo models of animal tissue, and also for use in live animals.

Reduced Oxidative Phosphorylation and Increased Glycolysis in Human Glaucoma Lamina Cribrosa Cells.

Purpose: The lamina cribrosa (LC) is a key site of damage in glaucomatous optic neuropathy. We previously found that glaucoma LC cells have an increased profibrotic gene expression, with mitochondrial dysfunction in the form of decreased mitochondrial membrane potential. Altered cell bioenergetics have recently been reported in organ fibrosis and in cancer. In this study, we carried out a systematic mitochondrial bioenergetic assessment and measured markers of alternative sources of cellular energy in normal and glaucoma LC cells. Methods: LC cells from three glaucoma donors and three age-matched normal controls were assessed using VICTOR X4 Perkin Elmer (Waltham, MA) plate reader with different phosphorescent and luminescent probes. adenosine triphosphate levels, oxygen consumption rate, and extracellular acidification were measured and normalized to total protein content. RNA and protein expression levels of MCT1, MCT4, MTFHD2, and GLS2 were quantified using real-time RT-PCR and Western blotting. Results: Glaucoma LC cells contain significantly less adenosine triphosphate (P < .05) when supplied with either glucose or galactose. They also showed significantly diminished oxygen consumption in both basal and maximal respiration with more lactic acid contribution in ECA. Both mRNA and protein expression levels of MCT1, MCT4, MTHFD2, and GLS2 were significantly increased in glaucoma LC cells. Conclusions: We demonstrate evidence of metabolic reprogramming (The Warburg effect) in glaucoma LC cells. Expression of markers of glycolysis, glutamine, and one carbon metabolism are elevated in glaucoma cells at both the mRNA and protein levels. A better understanding of bioenergetics in glaucoma may help in the development of new therapeutics.

Unusually efficient CUG initiation of an overlapping reading frame in POLG mRNA yields novel protein POLGARF.

While near-cognate codons are frequently used for translation initiation in eukaryotes, their efficiencies are usually low (<10% compared to an AUG in optimal context). Here, we describe a rare case of highly efficient near-cognate initiation. A CUG triplet located in the 5' leader of POLG messenger RNA (mRNA) initiates almost as efficiently (∼60 to 70%) as an AUG in optimal context. This CUG directs translation of a conserved 260-triplet-long overlapping open reading frame (ORF), which we call POLGARF (POLG Alternative Reading Frame). Translation of a short upstream ORF 5' of this CUG governs the ratio between POLG (the catalytic subunit of mitochondrial DNA polymerase) and POLGARF synthesized from a single POLG mRNA. Functional investigation of POLGARF suggests a role in extracellular signaling. While unprocessed POLGARF localizes to the nucleoli together with its interacting partner C1QBP, serum stimulation results in rapid cleavage and secretion of a POLGARF C-terminal fragment. Phylogenetic analysis shows that POLGARF evolved ∼160 million y ago due to a mammalian-wide interspersed repeat (MIR) transposition into the 5' leader sequence of the mammalian POLG gene, which became fixed in placental mammals. This discovery of POLGARF unveils a previously undescribed mechanism of de novo protein-coding gene evolution.

Correction to: Disruption of hypoxia-inducible fatty acid binding protein 7 induces beige fat-like differentiation and thermogenesis in breast cancer cells.

[This corrects the article DOI: 10.1186/s40170-020-00219-4.].

Disruption of hypoxia-inducible fatty acid binding protein 7 induces beige fat-like differentiation and thermogenesis in breast cancer cells.

BACKGROUND: Humans produce heat through non-shivering thermogenesis, a metabolic process that occurs in inducible beige adipocytes expressing uncoupling protein 1 (UCP1). UCP1 dissipates the proton gradient of the mitochondrial inner membrane and converts that energy into heat. It is unclear whether cancer cells can exhibit autonomous thermogenesis. Previously, we found that the knockdown of hypoxia-inducible fatty acid binding protein 7 (FABP7) increased reactive oxygen species (ROS) in breast cancer cells. ROS are known to induce beige adipocyte differentiation. METHODS: We investigated the association of tumor hypoxia, FABP7, and UCP1 across breast cancer patients using METABRIC and TCGA data sets. Furthermore, using a breast cancer cell line, HCC1806, we tested the effect of FABP7 knockdown on cellular physiology including thermogenesis. RESULTS: We found a strong mutual exclusivity of FABP7 and UCP1 expression both in METABRIC and in TCGA, indicating major metabolic phenotypic differences. FABP7 was preferentially distributed in poorly differentiated-, estrogen receptor (ER) negative tumors. In contrast, UCP1 was highly expressed in normal ducts and well-differentiated-, ER positive-, less hypoxic tumors. In the cell line-based experiments, UCP1 and its transcriptional regulators were upregulated upon FABP7 knockdown. UCP1 was induced in about 20% of cancer cells, and the effect was increased further in hypoxia. UCP1 depolarized mitochondrial membranes at the site of expression. UCP1 induction was associated with the increase in proton leak, glycolysis, and maximal respiration, mimicking the typical energy profile of beige adipocytes. Most importantly, UCP1 induction elevated cancer cell temperature associated with increased vulnerability to hypoxia and γ-irradiation. CONCLUSIONS: We demonstrated that breast cancer cells can undergo thermogenesis through UCP1 induction. Disrupting FABP7-mediated fatty acid metabolism can unlock UCP1-mediated thermogenesis, potentially making it possible to develop therapies to target thermogenesis. Further study would be warranted to investigate the effect of rise in temperature of cancer cells on patients' outcomes and the relationship to other metabolic pathways.

Visualization of Stem Cell Niche by Fluorescence Lifetime Imaging Microscopy.

Fluorescence lifetime imaging microscopy (FLIM), enabling live quantitative multiparametric analyses, is an emerging bioimaging approach in tissue engineering and regenerative medicine. When combined with stem cell-derived intestinal organoid models, FLIM allows for tracing stem cells and monitoring of their proliferation, metabolic fluxes, and oxygenation. It is compatible with the use of live Matrigel-grown intestinal organoids produced from primary adult stem cells, crypts, and transgenic Lgr5-GFP mice. In this chapter we summarize available experimental protocols, imaging platforms (one- and two-photon excited FLIM, phosphorescence lifetime imaging microscopy (PLIM)) and provide the anticipated data for FLIM imaging of the live intestinal organoids, focusing on labeling of cell proliferation, its colocalization with the stem cell niche, measured local oxygenation, autofluorescence, and some other parameters. The protocol is illustrated with examples of multiparameter imaging, employing spectral and "time domain"-based separation of dyes, probes, and assays.

Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context.

Eukaryotic translation initiation involves preinitiation ribosomal complex 5'-to-3' directional probing of mRNA for codons suitable for starting protein synthesis. The recognition of codons as starts depends on the codon identity and on its immediate nucleotide context known as Kozak context. When the context is weak (i.e., nonoptimal), leaky scanning takes place during which a fraction of ribosomes continues the mRNA probing. We explored the relationship between the context of AUG codons annotated as starts of protein-coding sequences and the next AUG codon occurrence. We found that AUG codons downstream from weak starts occur in the same frame more frequently than downstream from strong starts. We suggest that evolutionary selection on in-frame AUGs downstream from weak start codons is driven by the advantage of the reduction of wasteful out-of-frame product synthesis and also by the advantage of producing multiple proteoforms from certain mRNAs. We confirmed translation initiation downstream from weak start codons using ribosome profiling data. We also tested translation of alternative start codons in 10 specific human genes using reporter constructs. In all tested cases, initiation at downstream start codons was more productive than at the annotated ones. In most cases, optimization of Kozak context did not completely abolish downstream initiation, and in the specific example of CMPK1 mRNA, the optimized start remained unproductive. Collectively, our work reveals previously uncharacterized forces shaping the evolution of protein-coding genes and points to the plurality of translation initiation and the existence of sequence features influencing start codon selection, other than Kozak context.

A deeper understanding of intestinal organoid metabolism revealed by combining fluorescence lifetime imaging microscopy (FLIM) and extracellular flux analyses.

Stem cells and the niche in which they reside feature a complex microenvironment with tightly regulated homeostasis, cell-cell interactions and dynamic regulation of metabolism. A significant number of organoid models has been described over the last decade, yet few methodologies can enable single cell level resolution analysis of the stem cell niche metabolic demands, in real-time and without perturbing integrity. Here, we studied the redox metabolism of Lgr5-GFP intestinal organoids by two emerging microscopy approaches based on luminescence lifetime measurement - fluorescence-based FLIM for NAD(P)H, and phosphorescence-based PLIM for real-time oxygenation. We found that exposure of stem (Lgr5-GFP) and differentiated (no GFP) cells to high and low glucose concentrations resulted in measurable shifts in oxygenation and redox status. NAD(P)H-FLIM and O2-PLIM both indicated that at high 'basal' glucose conditions, Lgr5-GFP cells had lower activity of oxidative phosphorylation when compared with cells lacking Lgr5. However, when exposed to low (0.5 mM) glucose, stem cells utilized oxidative metabolism more dynamically than non-stem cells. The high heterogeneity of complex 3D architecture and energy production pathways of Lgr5-GFP organoids were also confirmed by the extracellular flux (XF) analysis. Our data reveals that combined analysis of NAD(P)H-FLIM and organoid oxygenation by PLIM represents promising approach for studying stem cell niche metabolism in a live readout.