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.

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.

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.

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.

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.

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.

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.

Estimation of the Mitochondrial Membrane Potential Using Fluorescence Lifetime Imaging Microscopy.

Monitoring of cell metabolism represents an important application area for fluorescence lifetime imaging microscopy (FLIM). In particular, assessment of mitochondrial membrane potential (MMP) in complex three-dimensional multicellular in vitro, ex vivo, and in vivo models would enable improved segmentation and functional discrimination of cell types, directly report on the mitochondrial function and complement the quenched-phosphorescence detection of cellular O2 and two-photon excited FLIM of endogenous NAD(P)H. Here, we report the green and orange-emitting fluorescent dyes SYTO and tetramethylrhodamine methyl ester (TMRM) as potential FLIM probes for MMP. In addition to nuclear, SYTO 16 and 24 dyes also display mitochondrial accumulation. FLIM with the culture of human colon cancer HCT116 cells allowed observation of the heterogeneity of mitochondrial polarization during the cell cycle progression. The dyes also demonstrated good performance with 3D cultures of Lgr5-GFP mouse intestinal organoids, providing efficient and quick cell staining and compatibility with two-photon excitation. Multiplexed imaging of Lgr5-GFP, proliferating cells (Hoechst 33342-aided FLIM), and TMRM-FLIM allowed us to identify the population of metabolically active cells in stem cell niche. TMRM-FLIM enabled to visualize the differences in membrane potential between Lgr5-positive and other proliferating and differentiated cell types. Altogether, SYTO 24 and TMRM dyes represent promising markers for advanced FLIM-based studies of cell bioenergetics with complex 3D and in vivo models. © 2019 International Society for Advancement of Cytometry.

Imaging of oxygen and hypoxia in cell and tissue samples.

Molecular oxygen (O2) is a key player in cell mitochondrial function, redox balance and oxidative stress, normal tissue function and many common disease states. Various chemical, physical and biological methods have been proposed for measurement, real-time monitoring and imaging of O2 concentration, state of decreased O2 (hypoxia) and related parameters in cells and tissue. Here, we review the established and emerging optical microscopy techniques allowing to visualize O2 levels in cells and tissue samples, mostly under in vitro and ex vivo, but also under in vivo settings. Particular examples include fluorescent hypoxia stains, fluorescent protein reporter systems, phosphorescent probes and nanosensors of different types. These techniques allow high-resolution mapping of O2 gradients in live or post-mortem tissue, in 2D or 3D, qualitatively or quantitatively. They enable control and monitoring of oxygenation conditions and their correlation with other biomarkers of cell and tissue function. Comparison of these techniques and corresponding imaging setups, their analytical capabilities and typical applications are given.

Insulin-like growth factor 1 signaling is essential for mitochondrial biogenesis and mitophagy in cancer cells.

Mitochondrial activity and metabolic reprogramming influence the phenotype of cancer cells and resistance to targeted therapy. We previously established that an insulin-like growth factor 1 (IGF-1)-inducible mitochondrial UTP carrier (PNC1/SLC25A33) promotes cell growth. This prompted us to investigate whether IGF signaling is essential for mitochondrial maintenance in cancer cells and whether this contributes to therapy resistance. Here we show that IGF-1 stimulates mitochondrial biogenesis in a range of cell lines. In MCF-7 and ZR75.1 breast cancer cells, IGF-1 induces peroxisome proliferator-activated receptor γ coactivator 1ß (PGC-1ß) and PGC-1α-related coactivator (PRC). Suppression of PGC-1ß and PRC with siRNA reverses the effects of IGF-1 and disrupts mitochondrial morphology and membrane potential. IGF-1 also induced expression of the redox regulator nuclear factor-erythroid-derived 2-like 2 (NFE2L2 alias NRF-2). Of note, MCF-7 cells with acquired resistance to an IGF-1 receptor (IGF-1R) tyrosine kinase inhibitor exhibited reduced expression of PGC-1ß, PRC, and mitochondrial biogenesis. Interestingly, these cells exhibited mitochondrial dysfunction, indicated by reactive oxygen species expression, reduced expression of the mitophagy mediators BNIP3 and BNIP3L, and impaired mitophagy. In agreement with this, IGF-1 robustly induced BNIP3 accumulation in mitochondria. Other active receptor tyrosine kinases could not compensate for reduced IGF-1R activity in mitochondrial protection, and MCF-7 cells with suppressed IGF-1R activity became highly dependent on glycolysis for survival. We conclude that IGF-1 signaling is essential for sustaining cancer cell viability by stimulating both mitochondrial biogenesis and turnover through BNIP3 induction. This core mitochondrial protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer.

Nanoparticle-based fluoroionophore for analysis of potassium ion dynamics in 3D tissue models and in vivo.

The imaging of real-time fluxes of K+ ions in live cell with high dynamic range (5-150 mM) is of paramount importance for neuroscience and physiology of the gastrointestinal tract, kidney and other tissues. In particular, the research on high-performance deep-red fluorescent nanoparticle-based biosensors is highly anticipated. We found that BODIPY-based FI3 K+-sensitive fluoroionophore encapsulated in cationic polymer RL100 nanoparticles displays unusually strong efficiency in staining of broad spectrum of cell models, such as primary neurons and intestinal organoids. Using comparison of brightness, photostability and fluorescence lifetime imaging microscopy (FLIM) we confirmed that FI3 nanoparticles display distinctively superior intracellular staining compared to the free dye. We evaluated FI3 nanoparticles in real-time live cell imaging and found that it is highly useful for monitoring intra- and extracellular K+ dynamics in cultured neurons. Proof-of-concept in vivo brain imaging confirmed applicability of the biosensor for visualization of epileptic seizures. Collectively, this data makes fluoroionophore FI3 a versatile cross-platform fluorescent biosensor, broadly compatible with diverse experimental models and that crown ether-based polymer nanoparticles can provide a new venue for design of efficient fluorescent probes.

Quantitative analysis of mucosal oxygenation using ex vivo imaging of healthy and inflamed mammalian colon tissue.

Colonic inflammation is associated with decreased tissue oxygenation, significantly affecting gut homeostasis. However, the crosstalk between O2 consumption and supply in the inflamed tissue are not fully understood. Using a murine model of colitis, we analysed O2 in freshly prepared samples of healthy and inflamed colon tissue. We developed protocols for efficient ex vivo staining of mouse distal colon mucosa with a cell-penetrating O2 sensitive probe Pt-Glc and high-resolution imaging of O2 concentration in live tissue by confocal phosphorescence lifetime-imaging microscopy (PLIM). Microscopy analysis revealed that Pt-Glc stained mostly the top 50-60 µm layer of the mucosa, with high phosphorescence intensity in epithelial cells. Measured O2 values in normal mouse tissue ranged between 5 and 35 µM (4-28 Torr), tending to decrease in the deeper tissue areas. Four-day treatment with dextran sulphate sodium (DSS) triggered colon inflammation, as evidenced by an increase in local IL6 and mKC mRNA levels, but did not affect the gross architecture of colonic epithelium. We further observed an increase in oxygenation, partial activation of hypoxia inducible factor (HIF) 1 signalling, and negative trends in pyruvate dehydrogenase activity and O2 consumption rate in the colitis mucosa, suggesting a decrease in mitochondrial respiration, which is known to be regulated via HIF-1 signalling and pyruvate oxidation rate. These results along with efficient staining with Pt-Glc of rat and human colonic mucosa reveal high potential of PLIM platform as a powerful tool for the high-resolution analysis of the intestinal tissue oxygenation in patients with inflammatory bowel disease and other pathologies, affecting tissue respiration.

Cellular ROS imaging with hydro-Cy3 dye is strongly influenced by mitochondrial membrane potential.

BACKGROUND: Hydrocyanines are widely used as fluorogenic probes to monitor reactive oxygen species (ROS) generation in cells. Their brightness, stability to autoxidation and photobleaching, large signal change upon oxidation, pH independence and red/near infrared emission are particularly attractive for imaging ROS in live tissue. METHODS: Using confocal fluorescence microscopy we have examined an interference of mitochondrial membrane potential (ΔΨm) with fluorescence intensity and localisation of a commercial hydro-Cy3 probe in respiring and non-respiring colon carcinoma HCT116 cells. RESULTS: We found that the oxidised (fluorescent) form of hydro-Cy3 is highly homologous to the common ΔΨm-sensitive probe JC-1, which accumulates and aggregates only in 'energised' negatively charged mitochondrial matrix. Therefore, hydro-Cy3 oxidised by hydroxyl and superoxide radicals tends to accumulate in mitochondrial matrix, but dissipates and loses brightness as soon as ΔΨm is compromised. Experiments with mitochondrial inhibitor oligomycin and uncoupler FCCP, as well as a common ROS producer paraquat demonstrated that signals of the oxidised hydro-Cy3 probe rapidly and strongly decrease upon mitochondrial depolarisation, regardless of the rate of cellular ROS production. CONCLUSIONS: While analysing ROS-derived fluorescence of commercial hydrocyanine probes, an accurate control of ΔΨm is required. GENERAL SIGNIFICANCE: If not accounted for, non-specific effect of mitochondrial polarisation state on the behaviour of oxidised hydrocyanines can cause artefacts and data misinterpretation in ROS studies.

The Ca2+/Mn2+-transporting SPCA2 pump is regulated by oxygen and cell density in colon cancer cells.

The mammalian SPCA1 and SPCA2 ATPases localize in membranes of the secretory pathway and transport ions of Ca(2+) and Mn(2+) The role of tissue-specific SPCA2 isoform, highly expressed in lungs, mammary gland and gastrointestinal tract, is poorly understood. To elucidate the function of SPCA2, we studied human colon cancer HCT116 cells, grown under ambient and decreased O2 levels. We found that in contrast with other Ca(2+)-ATPase isoforms the expression of SPCA2 was up-regulated under hypoxia (3% O2), in both adherent (2D) and spheroid (3D) cultures. In spheroids, experiencing lowest O2 levels (30-50 µM, measured by phosphorescence lifetime imaging microscopy), we observed lower staining with reactive oxygen species (ROS)-specific fluorescent probe, which correlated with increased SPCA2. However, SPCA2 expression was up-regulated in cells exposed to reactive oxygen and nitrogen species donors, and when grown at higher density. We noticed that the culture exposed to hypoxia showed overall increase in S phase-positive cells and hypothesized that SPCA2 up-regulation under hypoxia can be linked to Mn(2+)-dependent cell cycle arrest. Consequently, we found that SPCA2-transfected cells display a higher number of cells entering S phase. Altogether, our results point at the important role of SPCA2 in regulation of cell cycle in cancer cells.

Multi-Parametric Imaging of Hypoxia and Cell Cycle in Intestinal Organoid Culture.

Dynamics of oxygenation of tissue and stem cell niches are important for understanding physiological function of the intestine in normal and diseased states. Only a few techniques allow live visualization of tissue hypoxia at cellular level and in three dimensions. We describe an optimized protocol, which uses cell-penetrating O2-sensitive probe, Pt-Glc and phosphorescence lifetime imaging microscopy (PLIM), to analyze O2 distribution in mouse intestinal organoids. Unlike the other indirect and end-point hypoxia stains, or point measurements with microelectrodes, this method provides high-resolution real-time visualization of O2 in organoids. Multiplexing with conventional fluorescent live cell imaging probes such as the Hoechst 33342-based FLIM assay of cell proliferation, and immunofluorescence staining of endogenous proteins, allows analysis of key physiologic parameters under O2 control in organoids. The protocol is useful for gastroenterology and physiology of intestinal tissue, hypoxia research, regenerative medicine, studying host-microbiota interactions and bioenergetics.

A novel effect of DMOG on cell metabolism: direct inhibition of mitochondrial function precedes HIF target gene expression.

Abnormal accumulation of oncometabolite fumarate and succinate is associated with inhibition of mitochondrial function and carcinogenesis. By competing with α-ketoglutarate, oncometabolites also activate hypoxia inducible factors (HIFs), which makes oncometabolite mimetics broadly utilised in hypoxia research. We found that dimethyloxalylglycine (DMOG), a synthetic analogue of α-ketoglutarate, commonly used to induce HIF signalling, inhibits O2 consumption in cancer cell lines HCT116 and PC12, well before activation of HIF pathways. A rapid suppression of cellular respiration was accompanied by a decrease in histone H4 lysine 16 acetylation and not abolished by double knockdown of HIF-1α and HIF-2α. In agreement with this, production of NADH and state 3 respiration in isolated mitochondria were down-regulated by the de-esterified DMOG derivative, N-oxalylglycine. Exploring the roles of DMOG as a putative inhibitor of glutamine/α-ketoglutarate metabolic axis, we found that the observed suppression of OxPhos and compensatory activation of glycolytic ATP flux make cancer cells vulnerable to combined treatment with DMOG and inhibitors of glycolysis. On the other hand, DMOG treatment impairs deep cell deoxygenation in 3D tissue culture models, demonstrating a potential to relieve functional stress imposed by deep hypoxia on tumour, ischemic or inflamed tissues. Indeed, using a murine model of colitis, we found that O2 availability in the inflamed colon tissue rapidly increased after application of DMOG, which could contribute to the known therapeutic effect of this compound. Overall, our results provide new insights into the relationship between mitochondrial function, O2 availability, metabolic reprogramming and associated diseases.

Sulforhodamine Nanothermometer for Multiparametric Fluorescence Lifetime Imaging Microscopy.

Live cells function within narrow limits of physiological temperature (T) and O2 and metabolite concentrations. We have designed a cell-permeable T-sensitive fluorescence lifetime-based nanoprobe based on lipophilic sulforhodamine, which stains 2D and 3D cell models, shows cytoplasmic localization, and has a robust response to T (∼0.037 ns/K). Subsequently, we evaluated the probe and fluorescence lifetime imaging microscopy (FLIM) technique for combined imaging of T and O2 gradients in metabolically active cells. We found that in adherent 2D culture of HCT116 cells intracellular T and O2 are close to ambient values. However, in 3D spheroid structures having size >200 µm, T and O2 gradients become pronounced. These microgradients can be enhanced by treatment with mitochondrial uncouplers or dissipated by drug-induced disaggregation of the spheroids. Thus, we demonstrate the existence of local microgradients of T in 3D cell models and utility of combined imaging of O2 and T.

Metallochelate Coupling of Phosphorescent Pt-Porphyrins to Peptides, Proteins, and Self-Assembling Protein Nanoparticles.

Specific and reversible metallochelate coupling via nitrilotriacetate (NTA) moiety is widely used for immobilization, purification, and labeling of oligo(histidine)-tagged proteins. Here, we evaluated this strategy to label various peptides and proteins with phosphorescent Pt-porphyrin derivatives bearing NTA group(s). Zn(2+) complexes were shown to have minimal effect on the photophysics of the porphyrin moiety, allowing quenched-phosphorescence sensing of O2. We complexed the PtTFPP-NTA conjugate with His-containing peptide that can facilitate intracellular loading, and observed efficient accumulation and phosphorescent staining of MEF cells. The more hydrophilic PtCP-NTA conjugate was also seen to form stable complexes with larger polypeptide constructs based on fluorescent proteins, and with subunits of protein nanoparticles, which retained their ability to self-assemble. Testing in phosphorescence lifetime based O2 sensing assays on a fluorescence reader and PLIM microscope revealed that phosphorescent metallochelate complexes perform similarly to the existing O2 probes. Thus, metallochelate coupling allows simple preparation of different types of biomaterials labeled with phosphorescent Pt-porphyrins.

Differential contribution of key metabolic substrates and cellular oxygen in HIF signalling.

Changes in availability and utilisation of O2 and metabolic substrates are common in ischemia and cancer. We examined effects of substrate deprivation on HIF signalling in PC12 cells exposed to different atmospheric O2. Upon 2-4h moderate hypoxia, HIF-α protein levels were dictated by the availability of glutamine and glucose, essential for deep cell deoxygenation and glycolytic ATP flux. Nuclear accumulation of HIF-1α dramatically decreased upon inhibition of glutaminolysis or glutamine deprivation. Elevation of HIF-2α levels was transcription-independent and associated with the activation of Akt and Erk1/2. Upon 2h anoxia, HIF-2α levels strongly correlated with cellular ATP, produced exclusively via glycolysis. Without glucose, HIF signalling was suppressed, giving way to other regulators of cell adaptation to energy crisis, e.g. AMPK. Consequently, viability of cells deprived of O2 and glucose decreased upon inhibition of AMPK with dorsomorphin. The capacity of cells to accumulate HIF-2α decreased after 24h glucose deprivation. This effect, associated with increased AMPKα phosphorylation, was sensitive to dorsomorphin. In chronically hypoxic cells, glutamine played no major role in HIF-2α accumulation, which became mainly glucose-dependent. Overall, the availability of O2 and metabolic substrates intricately regulates HIF signalling by affecting cell oxygenation, ATP levels and pathways involved in production of HIF-α.