Publications by authors named "Alexander V Zhdanov"

39 Publications

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

Sci Rep 2020 11 4;10(1):19006. Epub 2020 Nov 4.

School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland.

O 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 O 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 O in the tissue within 20 s. Inhibition of tissue respiration or limitation of O diffusion to tissue produced the anticipated increases or decreases in O levels, respectively. The difference in O 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 O 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.
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http://dx.doi.org/10.1038/s41598-020-75928-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642408PMC
November 2020

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

Invest Ophthalmol Vis Sci 2020 11;61(13)

Department of Ophthalmology, Mater Misericordiae University Hospital, Dublin, Ireland.

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.
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http://dx.doi.org/10.1167/iovs.61.13.4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645202PMC
November 2020

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

Proc Natl Acad Sci U S A 2020 10 21;117(40):24936-24946. Epub 2020 Sep 21.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia, 119992;

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 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 ( 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 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 evolved ∼160 million y ago due to a mammalian-wide interspersed repeat (MIR) transposition into the 5' leader sequence of the mammalian gene, which became fixed in placental mammals. This discovery of unveils a previously undescribed mechanism of de novo protein-coding gene evolution.
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http://dx.doi.org/10.1073/pnas.2001433117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547235PMC
October 2020

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

Genome Res 2020 Jul 15;30(7):974-984. Epub 2020 Jul 15.

School of Biochemistry and Cell Biology, University College Cork, Cork, T12 XF62 Ireland.

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 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.
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http://dx.doi.org/10.1101/gr.257352.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397870PMC
July 2020

Short-chain fatty acids and microbiota metabolites attenuate ghrelin receptor signaling.

FASEB J 2019 12 9;33(12):13546-13559. Epub 2019 Oct 9.

Alimentary Pharmabiotic Centre (APC) Microbiome Ireland, University College Cork, Cork, Ireland.

The gastrointestinal microbiota is emerging as a unique and inexhaustible source for metabolites with potential to modulate G-protein coupled receptors (GPCRs). The ghrelin receptor [growth hormone secretagogue receptor (GHSR)-1a] is a GPCR expressed throughout both the gut and the brain and plays a crucial role in maintaining energy balance, metabolism, and the central modulation of food intake, motivation, reward, and mood. To date, few studies have investigated the potential of the gastrointestinal microbiota and its metabolites to modulate GPCR signaling. Here we investigate the ability of short-chain fatty acids (SCFAs), lactate, and different bacterial strains, including and genera, to modulate GHSR-1a signaling. We identify, for what is to our knowledge the first time, a potent effect of microbiota-derived metabolites on GHSR-1a signaling with potential significant consequences for host metabolism and physiology. We show that SCFAs, lactate, and bacterial supernatants are able to attenuate ghrelin-mediated signaling through the GHSR-1a. We suggest a novel route of communication between the gut microbiota and the host modulation of GHSR-1a receptor signaling. Together, this highlights the emerging therapeutic potential in the exploration of the microbiota metabolome in the specific targeting of key GPCRs, with pleiotropic actions that span both the CNS and periphery.-Torres-Fuentes, C., Golubeva, A. V., Zhdanov, A. V., Wallace, S., Arboleya, S., Papkovsky, D. B., El Aidy, S., Ross, P., Roy, B. L., Stanton, C., Dinan, T. G., Cryan, J. F., Schellekens, H. Short-chain fatty acids and microbiota metabolites attenuate ghrelin receptor signaling.
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http://dx.doi.org/10.1096/fj.201901433RDOI Listing
December 2019

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

Adv Funct Mater 2018 Feb 5;28(9). Epub 2018 Jan 5.

ABCRF, School of Biochemistry and Cell biology, University College Cork, Cork, Ireland.

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 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.
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http://dx.doi.org/10.1002/adfm.201704598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6157274PMC
February 2018

AMD1 mRNA employs ribosome stalling as a mechanism for molecular memory formation.

Nature 2018 01 3;553(7688):356-360. Epub 2018 Jan 3.

School of Biochemistry and Cell Biology, University College Cork, Cork T12 YN60, Ireland.

In addition to acting as template for protein synthesis, messenger RNA (mRNA) often contains sensory sequence elements that regulate this process. Here we report a new mechanism that limits the number of complete protein molecules that can be synthesized from a single mRNA molecule of the human AMD1 gene encoding adenosylmethionine decarboxylase 1 (AdoMetDC). A small proportion of ribosomes translating AMD1 mRNA stochastically read through the stop codon of the main coding region. These readthrough ribosomes then stall close to the next in-frame stop codon, eventually forming a ribosome queue, the length of which is proportional to the number of AdoMetDC molecules that were synthesized from the same AMD1 mRNA. Once the entire spacer region between the two stop codons is filled with queueing ribosomes, the queue impinges upon the main AMD1 coding region halting its translation. Phylogenetic analysis suggests that this mechanism is highly conserved in vertebrates and existed in their common ancestor. We propose that this mechanism is used to count and limit the number of protein molecules that can be synthesized from a single mRNA template. It could serve to safeguard from dysregulated translation that may occur owing to errors in transcription or mRNA damage.
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http://dx.doi.org/10.1038/nature25174DOI Listing
January 2018

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

J Biol Chem 2017 10 18;292(41):16983-16998. Epub 2017 Aug 18.

From the Cell Biology Laboratory and

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 1β (PGC-1β) and PGC-1α-related coactivator (PRC). Suppression of PGC-1β 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-1β, 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.
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http://dx.doi.org/10.1074/jbc.M117.792838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641874PMC
October 2017

Low energy costs of F1Fo ATP synthase reversal in colon carcinoma cells deficient in mitochondrial complex IV.

Free Radic Biol Med 2017 05 9;106:184-195. Epub 2017 Feb 9.

School of Biochemistry & Cell Biology, University College Cork, Cork, Ireland.

Mitochondrial polarisation is paramount for a variety of cellular functions. Under ischemia, mitochondrial membrane potential (ΔΨm) and proton gradient (ΔpH) are maintained via a reversal of mitochondrial F1Fo ATP synthase (mATPase), which can rapidly deplete ATP and drive cells into energy crisis. We found that under normal conditions in cells with disassembled cytochrome c oxidase complex (COX-deficient HCT116), mATPase maintains ΔΨm at levels only 15-20% lower than in WT cells, and for this utilises relatively little ATP. For a small energy expenditure, mATPase enables mitochondrial ΔpH, protein import, Ca turnover, and supports free radical detoxication machinery enlarged to protect the cells from oxidative damage. Whereas in COX-deficient cells the main source of ATP is glycolysis, the ΔΨm is still maintained upon inhibition of the adenine nucleotide translocators with bongkrekic acid and carboxyatractyloside, indicating that the role of ANTs is redundant, and matrix substrate level phosphorylation alone or in cooperation with ATP-Mg/P carriers can continuously support the mATPase activity. Intriguingly, we found that mitochondrial complex III is active, and it contributes not only to free radical production, but also to ΔΨm maintenance and energy budget of COX-deficient cells. Overall, this study demonstrates that F1Fo ATP synthase can support general mitochondrial and cellular functions, working in extremely efficient 'energy saving' reverse mode and flexibly recruiting free radical detoxication and ATP producing / transporting pathways.
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http://dx.doi.org/10.1016/j.freeradbiomed.2017.02.025DOI Listing
May 2017

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

Biochim Biophys Acta Gen Subj 2017 Feb 4;1861(2):198-204. Epub 2016 Nov 4.

School of Biochemistry & Cell Biology, University College Cork, Cork, Ireland.

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.
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http://dx.doi.org/10.1016/j.bbagen.2016.10.023DOI Listing
February 2017

Phosphorescence based O sensors - Essential tools for monitoring cell and tissue oxygenation and its impact on metabolism.

Free Radic Biol Med 2016 12 24;101:202-210. Epub 2016 Oct 24.

School of Biochemistry and Cell Biology, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Ireland.

Oxygenation condition at the cellular level is a critical factor in tissue physiology and common pathophysiological states including cancer, metabolic disorders, ischemia-reperfusion injury and inflammation. O and ROS signalling and hypoxia research are rapidly growing areas spanning life and biomedical sciences, but still many current cell and tissue models and experimental set ups lack physiological relevance, particularly precise control of cellular O. Quenched-phosphorescence O sensing enables implementation of such in situ control of cellular O and the creation of physiological conditions in respiring samples analysed in vitro. The advantages of optical O sensing are the non-invasive, contactless, real-time, quantitative monitoring of O concentration, which can be performed in the gas or liquid phase, macroscopically or microscopically, by point measurement or in imaging mode, with sub-cellular spatial resolution, in a flexible manner and with various cell and tissue models. Significantly, this same technology can also be used to probe the metabolism of cells and tissue under specific oxygenation conditions and their responses to changing conditions. Here we describe the range of available O sensing systems and tools, their analytical capabilities, uses in cell/tissue physiology and hypoxia research, and strategies for integration in routine experimental procedures.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.09.018DOI Listing
December 2016

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

Cell Mol Life Sci 2017 01 10;74(1):141-151. Epub 2016 Aug 10.

School of Biochemistry and Cell Biology, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Ireland.

Colonic inflammation is associated with decreased tissue oxygenation, significantly affecting gut homeostasis. However, the crosstalk between O consumption and supply in the inflamed tissue are not fully understood. Using a murine model of colitis, we analysed O 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 O sensitive probe Pt-Glc and high-resolution imaging of O 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 O 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 O 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.
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http://dx.doi.org/10.1007/s00018-016-2323-xDOI Listing
January 2017

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

Biochim Biophys Acta 2015 Oct 2;1847(10):1254-66. Epub 2015 Jul 2.

School of Biochemistry & Cell Biology, University College Cork, Cork, Ireland.

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.
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http://dx.doi.org/10.1016/j.bbabio.2015.06.016DOI Listing
October 2015

Prenatal stress-induced alterations in major physiological systems correlate with gut microbiota composition in adulthood.

Psychoneuroendocrinology 2015 Oct 17;60:58-74. Epub 2015 Jun 17.

Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland; Department of Anatomy & Neuroscience, University College Cork, Cork, Ireland. Electronic address:

Early-life adverse experiences, including prenatal stress (PNS), are associated with a higher prevalence of neurodevelopmental, cardiovascular and metabolic disorders in affected offspring. Here, in a rat model of chronic PNS, we investigate the impact of late gestational stress on physiological outcomes in adulthood. Sprague-Dawley pregnant dams were subjected to repeated restraint stress from embryonic day 14 to day 20, and their male offspring were assessed at 4 months of age. PNS induced an exaggeration of the hypothalamic-pituitary-adrenal (HPA) axis response to stress, as well as an elevation of blood pressure and impairment of cognitive function. Altered respiratory control was also observed, as demonstrated by increased variability in basal respiratory frequency and abnormal frequency responses to both hypoxic and hypercapnic challenges. PNS also affected gastrointestinal neurodevelopment and function, as measured by a decrease in the innervation density of distal colon and an increase in the colonic secretory response to catecholaminergic stimulation. Finally, PNS induced long lasting alterations in the intestinal microbiota composition. 16S rRNA gene 454 pyrosequencing revealed a strong trend towards decreased numbers of bacteria in the Lactobacillus genus, accompanied by elevated abundance of the Oscillibacter, Anaerotruncus and Peptococcus genera in PNS animals. Strikingly, relative abundance of distinct bacteria genera significantly correlated with certain respiratory parameters and the responsiveness of the HPA axis to stress. Together, these findings provide novel evidence that PNS induces long-term maladaptive alterations in the gastrointestinal and respiratory systems, accompanied by hyper-responsiveness to stress and alterations in the gut microbiota.
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http://dx.doi.org/10.1016/j.psyneuen.2015.06.002DOI Listing
October 2015

Oxygen and glucose deprivation induces widespread alterations in mRNA translation within 20 minutes.

Genome Biol 2015 May 6;16:90. Epub 2015 May 6.

School of Biochemistry and Cell Biology, Western Gateway Building, University College Cork, Cork, Ireland.

Background: Oxygen and glucose metabolism play pivotal roles in many (patho)physiological conditions. In particular, oxygen and glucose deprivation (OGD) during ischemia and stroke results in extensive tissue injury and cell death.

Results: Using time-resolved ribosome profiling, we assess gene expression levels in a neural cell line, PC12, during the first hour of OGD. The most substantial alterations are seen to occur within the first 20 minutes of OGD. While transcription of only 100 genes is significantly altered during one hour of OGD, the translation response affects approximately 3,000 genes. This response involves reprogramming of initiation and elongation rates, as well as the stringency of start codon recognition. Genes involved in oxidative phosphorylation are most affected. Detailed analysis of ribosome profiles reveals salient alterations of ribosome densities on individual mRNAs. The mRNA-specific alterations include increased translation of upstream open reading frames, site-specific ribosome pauses, and production of alternative protein isoforms with amino-terminal extensions. Detailed analysis of ribosomal profiles also reveals six mRNAs with translated ORFs occurring downstream of annotated coding regions and two examples of dual coding mRNAs, where two protein products are translated from the same long segment of mRNA, but in two different frames.

Conclusions: These findings uncover novel regulatory mechanisms of translational response to OGD in mammalian cells that are different from the classical pathways such as hypoxia inducible factor (HIF) signaling, while also revealing sophisticated organization of protein coding information in certain genes.
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http://dx.doi.org/10.1186/s13059-015-0651-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4419486PMC
May 2015

Cell energy budget platform for assessment of cell metabolism.

Methods Mol Biol 2015 ;1265:333-48

School of Biochemistry and Cell Biology, University College Cork, Cavanagh Pharmacy Building, Office 1.28, College Road, Cork, Ireland,

Changes in bioenergetic parameters report on metabolic rearrangement, dysfunction of major pathways, and regulatory processes within the cell, adaptation to energy stress, or new physiological condition. A combined measurement of oxidative phosphorylation, glycolytic flux, the Krebs cycle activity, ATP levels, and total biomass allows detailed metabolic assessment. We describe a simple methodology for high-throughput multiparametric assessment of cell bioenergetics, called cell energy budget (CEB) platform, and demonstrate its practical use with cell models. The CEB relies on a standard multi-label reader with time-resolved fluorescence capabilities, the lanthanide probe pH-Xtra™ to measure extracellular acidification (ECA) associated with lactate (L-ECA) and combined lactate/CO2 (T-ECA) extrusion, the phosphorescent probe MitoXpress®-Xtra to measure oxygen consumption rate (OCR), the bioluminescent total ATP assay, and absorbance-based total protein assay. This approach can be further extended with the measurement of other cellular parameters, such as NAD(P)H, Ca(2+), mitochondrial pH, membrane potential, and redox state, using the corresponding fluorescent or luminescent probes.
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http://dx.doi.org/10.1007/978-1-4939-2288-8_23DOI Listing
October 2015

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

Exp Cell Res 2015 Jan 15;330(1):13-28. Epub 2014 Oct 15.

School of Biochemistry and Cell Biology, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Ireland.

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.
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http://dx.doi.org/10.1016/j.yexcr.2014.10.005DOI Listing
January 2015

Imaging oxygen in neural cell and tissue models by means of anionic cell-permeable phosphorescent nanoparticles.

Cell Mol Life Sci 2015 Jan 9;72(2):367-81. Epub 2014 Jul 9.

School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland,

Cell-permeable phosphorescent probes enable the study of cell and tissue oxygenation, bioenergetics, metabolism, and pathological states such as stroke and hypoxia. A number of such probes have been described in recent years, the majority consisting of cationic small molecule and nanoparticle structures. While these probes continue to advance, adequate staining for the study of certain cell types using live imaging techniques remains elusive; this is particularly true for neural cells. Here we introduce novel probes for the analysis of neural cells and tissues: negatively charged poly(methyl methacrylate-co-methacrylic acid)-based nanoparticles impregnated with a phosphorescent Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye (this form is referred to as PA1), and with an additional reference/antennae dye poly(9,9-diheptylfluorene-alt-9,9-di-p-tolyl-9H-fluorene) (this form is referred to as PA2). PA1 and PA2 are internalised by endocytosis, result in efficient staining in primary neurons, astrocytes, and PC12 cells and multi-cellular aggregates, and allow for the monitoring of local O(2) levels on a time-resolved fluorescence plate reader and PLIM microscope. PA2 also efficiently stains rat brain slices and permits detailed O(2) imaging experiments using both one and two-photon intensity-based modes and PLIM modes. Multiplexed analysis of embryonic rat brain slices reveals age-dependent staining patterns for PA2 and a highly heterogeneous distribution of O(2) in tissues, which we relate to the localisation of specific progenitor cell populations. Overall, these anionic probes are useful for sensing O(2) levels in various cells and tissues, particularly in neural cells, and facilitate high-resolution imaging of O(2) in 3D tissue models.
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http://dx.doi.org/10.1007/s00018-014-1673-5DOI Listing
January 2015

Small molecule phosphorescent probes for O imaging in 3D tissue models.

Biomater Sci 2014 Jun 28;2(6):853-866. Epub 2014 Jan 28.

Biochemistry Department, University College Cork, Cork, Ireland.

Monitoring of oxygenation is important for physiological experiments investigating the growth, differentiation and function of individual cells in 3D tissue models. Phosphorescence based O sensing and imaging potentially allow this task; however, current probes do not provide the desired bio-distribution and analytical performance. We present several new cell-penetrating phosphorescent conjugates of a Pt(ii)-tetrakis(pentafluorophenyl)porphine (PtPFPP) dye produced by click-modification with thiols, and perform their evaluation as O imaging probes for 3D tissue models. The hydrophilic glucose (Pt-Glc) and galactose (Pt-Gal) conjugates demonstrated minimal aggregation and self-quenching in aqueous media, and efficient in-depth staining of different cell types and multi-cellular aggregates at working concentrations ≤10 μM. The Pt-Glc probe was applied in high-resolution phosphorescence lifetime based O imaging (PLIM) in multi-cellular spheroids of cancer cells (PC12), primary neural cells (neurospheres) and slices of brain tissue, where it showed good analytical performance, minimal effects on cell viability and appropriate responses to O with phosphorescence lifetimes changing from 20 μs in air-saturated to 57 μs under deoxygenated conditions. In contrast, mono- and tetra-substituted oligoarginine conjugates of PtPFPP showed marked aggregation and unstable photophysical properties precluding their use as O sensing probes.
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http://dx.doi.org/10.1039/c3bm60272aDOI Listing
June 2014

Kinetic analysis of local oxygenation and respiratory responses of mammalian cells using intracellular oxygen-sensitive probes and time-resolved fluorometry.

Methods Enzymol 2014 ;542:183-207

Biochemistry Department, University College Cork, Cork, Ireland. Electronic address:

The cell-penetrating O2-sensing probes based on phosphorescent Pt-porphyrins provide a means to accurately measure O2 within cultured cells in a contact-less fashion. Via this approach, in situ oxygenation and respiratory responses can be monitored in real time on large number of cellular samples seeded in standard microtiter plates, by means of a commercial time-resolved fluorescence reader. Using neuronal PC12 cells as a model, here we describe the general strategy behind the use of phosphorescent Pt-porphyrins to measure O2 within cultured cells as well as detailed protocols to implement this experimental approach, including cell preparation and probe loading, instrument and plate setup, system calibration, and data processing. Two main protocols are presented: (1) a basic setup and optimization for the measurement of intracellular O2 under ambient O2 concentration; (2) the calibration of the system required to perform measurements under low O2 concentrations (hypoxia). A case study on the effects of bafilomycin A1 on PC12 cell function is included, illustrating how these methods can be combined with conventional bioenergetic markers to delineate how chemical and/or physical stimuli affect the metabolism of normal and malignant cells.
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http://dx.doi.org/10.1016/B978-0-12-416618-9.00010-8DOI Listing
May 2015

The mouse cyclophosphamide model of bladder pain syndrome: tissue characterization, immune profiling, and relationship to metabotropic glutamate receptors.

Physiol Rep 2014 27;2(3):e00260. Epub 2014 Mar 27.

Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland.

Abstract Painful bladder syndrome/Interstitial cystitis (PBS/IC) is a chronic disorder characterized clinically by recurring episodes of pelvic pain and increased urination frequency, significantly impairing patients' quality of life. Despite this, there is an unmet medical need in terms of effective diagnostics and treatment. Animal models are crucial in this endeavor. Systemic chronic administration of cyclophosphamide (CYP) in mice has been proposed as a relevant preclinical model of chronic bladder pain. However, molecular mechanisms underlying the pathogenesis of this model are lacking. Here, we show that mice, subjected to repetitive systemic injections of CYP, developed mild inflammatory response in bladder tissue characterized by submucosal edema, moderate increase in proinflammatory cytokine gene expression, and mastocytosis. No signs of massive inflammatory infiltrate, tissue hemorrhages, mucosal ulcerations and urothelium loss were observed. Instead, CYP treatment induced urothelium hyperplasia, accompanied by activation of proliferative signaling cascades, and a decrease in the expression of urothelium-specific markers. Metabotropic glutamate (mGlu) receptors have been implicated in chronic pain disorders. CYP administration induced differential changes in mGlu receptors mRNA levels in bladder tissue, without affecting gene expression at spinal cord level, pointing to the potential link between peripheral mGlu receptors and inflammation-induced bladder malfunction and hyperalgesia. Taken together, these data indicate that chronic CYP treatment in mice is a model of PBS mostly relevant to the major, nonulcerative subtype of the syndrome, characterized by a relatively unaltered mucosa and a sparse inflammatory response. This model can help to elucidate the pathogenetic mechanisms of the disease.
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http://dx.doi.org/10.1002/phy2.260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4002240PMC
April 2014

Imaging of neurosphere oxygenation with phosphorescent probes.

Biomaterials 2013 Dec 6;34(37):9307-17. Epub 2013 Sep 6.

Biochemistry Department, University College Cork, Cork, Ireland. Electronic address:

Multicellular spheroids are useful models of mammalian tissue for studies of cell proliferation, differentiation, replacement therapies and drug action. Having a size of 100-500 μm they mimic in vivo micro-environment and characteristic gradients of O2, pH and nutrients. We describe the use of cell-penetrating O2 probes based on phosphorescent Pt-porphyrins to perform high-resolution 2D and 3D mapping of O2 in spheroid structures by live cell fluorescence imaging technique. Optimised procedures for preparation of neurospheres from cortical neural cells isolated from embryonic rat brain, their staining with the phosphorescent O2 probes NanO2 and MM2 and subsequent analysis of oxygenation on different live cell imaging platforms, including widefield and confocal phosphorescence lifetime imaging microscopy (PLIM), conventional confocal and two-photon ratiometric intensity based O2 detection are presented. This is followed by a series of physiological experiments in which oxygenation patterns of the neurospheres are correlated with culturing conditions (atmospheric hypoxia and hyperoxia, size, growth factors), distribution of stem cells, mature neurons and astrocytes, HIF-2α stabilisation and responses to metabolic stimulation. The O2 imaging method allows multiplexing with many conventional fluorescent probes to perform multi-parametric imaging analysis of cells in 3D microenvironment. It can be applied to other types of spheroids and 3D tissue models.
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http://dx.doi.org/10.1016/j.biomaterials.2013.08.065DOI Listing
December 2013

Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling.

Biochim Biophys Acta 2014 Jan 23;1837(1):51-62. Epub 2013 Jul 23.

Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Ireland. Electronic address:

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O2 consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca(2+) or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment.
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http://dx.doi.org/10.1016/j.bbabio.2013.07.008DOI Listing
January 2014

Chronic hypoxia leads to a glycolytic phenotype and suppressed HIF-2 signaling in PC12 cells.

Biochim Biophys Acta 2013 Jun 24;1830(6):3553-69. Epub 2013 Feb 24.

Biochemistry Department, University College Cork, Cork, Ireland.

Background: Along with other regulators of cell metabolism, hypoxia-inducible factors HIF-1 and HIF-2 differentially regulate cell adaptation to hypoxia. Switches in HIF-1/HIF-2 signaling in chronic hypoxia have not been fully investigated.

Methods: Proliferation, viability, apoptosis, neuronal and bioenergetic markers, mitochondrial function, respiration, glycolysis, HIF signalling, responses to O2 and glucose deprivation (OGD) were examined using tumor PC12 and SH-SY5Y cells continuously grown at 3% O2.

Results: Hypoxic PC12 cells (H-cells) exhibit reduced proliferation and histone H4 acetylation, NGF-independent differentiation, activation of AMPK, inhibition of Akt, altered mitochondria and response to NGF. Cellular cytochrome c is increased with no effect on apoptosis. Reduction in respiration has minor effect on cellular ATP which is maintained through activated uptake (GLUT1) and utilization (HK2, PFK2) of glucose. H-cells exhibit resistance to OGD linked to increased glycogen stores. HIF-2alpha protein is decreased without changes in mRNA. Unlike HIF-1alpha, HIF-2alpha is not stabilized pharmacologically or by O2 deprivation. Capacity for HIF-2alpha stabilization is partly restored when H-cells are cultured at normoxia. In low-respiring SH-SY5Y cells cultured under the same conditions HIF-2alpha stabilization and energy budget are not affected.

Conclusions: In chronically hypoxic PC12 cells glycolytic energy budget, increased energy preservation and low susceptibility to OGD are observed. HIF-2alpha no longer orchestrates adaptive responses to anoxia.

General Significance: Demonstrated switch in HIF-1/HIF-2 signaling upon chronic hypoxia can facilitate cell survival in energy crisis, by regulating balance between energy saving and decrease in proliferation, on one hand and active cell growth and tumor expansion, on the other.
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http://dx.doi.org/10.1016/j.bbagen.2013.02.016DOI Listing
June 2013

Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling.

Biosci Rep 2012 Dec;32(6):587-95

Biochemistry Department, University College Cork, Cork, Ireland.

Mitochondrial uncoupling is implicated in many patho(physiological) states. Using confocal live cell imaging and an optical O2 sensing technique, we show that moderate uncoupling of the mitochondria with plecomacrolide Baf (bafilomycin A1) causes partial depolarization of the mitochondria and deep sustained deoxygenation of human colon cancer HCT116 cells subjected to 6% atmospheric O2. A decrease in iO2 (intracellular O2) to 0-10 μM, induced by Baf, is sufficient for stabilization of HIFs (hypoxia inducible factors) HIF-1α and HIF-2α, coupled with an increased expression of target genes including GLUT1 (glucose transporter 1), HIF PHD2 (prolyl hydroxylase domain 2) and CAIX (carbonic anhydrase IX). Under the same hypoxic conditions, treatment with Baf causes neither decrease in iO2 nor HIF-α stabilization in the low-respiring HCT116 cells deficient in COX (cytochrome c-oxidase). Both cell types display equal capacities for HIF-α stabilization by hypoxia mimetics DMOG (dimethyloxalylglycine) and CoCl2, thus suggesting that the effect of Baf under hypoxia is driven mainly by mitochondrial respiration. Altogether, by activating HIF signalling under moderate hypoxia, mitochondrial uncoupling can play an important regulatory role in colon cancer metabolism and modulate adaptation of cancer cells to natural hypoxic environments.
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http://dx.doi.org/10.1042/BSR20120085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497721PMC
December 2012

Assessment of cellular oxygen gradients with a panel of phosphorescent oxygen-sensitive probes.

Anal Chem 2012 Mar 29;84(6):2930-8. Epub 2012 Feb 29.

Biochemistry Department, University College Cork, Cork, Ireland.

The supply of oxygen (O(2)) to respiring tissue, cells, and mitochondria regulates metabolism, gene expression, and cell fate. Depending on the cell type and mitochondrial function, O(2) gradients between extra- and intracellular compartments may vary and play important physiological roles such as the regulation of activity of prolyl hydroxylases and adaptive responses to hypoxia. Here we present a new methodology for the analysis of localized O(2) gradients in cultures of adherent cells, using three phosphorescent Pt-porphyrin based probes with different localization. One new O(2) probe targeted to the cell membrane was developed and used together with existing MitoXpress and Nano2 probes to monitor mean pericellular (PC), extracellular (EC), and intracellular (IC) O(2) concentrations, respectively. Mouse fibroblasts and neuronal PC12 cells cultured in standard microplates were stained with probes and measured on a commercial time-resolved fluorescence reader in phosphorescence lifetime mode. Respiring cells exposed to various levels of atmospheric O(2) showed differences in oxygenation of their IC, PC, and EC compartments. Experiments with different cell numbers and modulation of respiration activity demonstrated that these gradients are dynamic and regulated by the O(2) diffusion and consumption rate. The new method facilitates the assessment of such gradients.
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http://dx.doi.org/10.1021/ac3000144DOI Listing
March 2012

Intracellular O2 sensing probe based on cell-penetrating phosphorescent nanoparticles.

ACS Nano 2011 Jul 20;5(7):5499-508. Epub 2011 Jun 20.

Biochemistry Department, University College Cork, Cavanagh Building, College Road, Cork, Ireland.

A new intracellular O(2) (icO(2)) sensing probe is presented, which comprises a nanoparticle (NP) formulation of a cationic polymer Eudragit RL-100 and a hydrophobic phosphorescent dye Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP). Using the time-resolved fluorescence (TR-F) plate reader set-up, cell loading was investigated in detail, particularly the effects of probe concentration, loading time, serum content in the medium, cell type, density, etc. The use of a fluorescent analogue of the probe in conjunction with confocal microscopy and flow cytometry analysis, revealed that cellular uptake of the NPs is driven by nonspecific energy-dependent endocytosis and that the probe localizes inside the cell close to the nucleus. Probe calibration in biological environment was performed, which allowed conversion of measured phosphorescence lifetime signals into icO(2) concentration (μM). Its analytical performance in icO(2) sensing experiments was demonstrated by monitoring metabolic responses of mouse embryonic fibroblast cells under ambient and hypoxic macroenvironment. The NP probe was seen to generate stable and reproducible signals in different types of mammalian cells and robust responses to their metabolic stimulation, thus allowing accurate quantitative analysis. High brightness and photostability allow its use in screening experiments with cell populations on a commercial TR-F reader, and for single cell analysis on a fluorescent microscope.
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http://dx.doi.org/10.1021/nn200807gDOI Listing
July 2011

Bactenecin 7 peptide fragment as a tool for intracellular delivery of a phosphorescent oxygen sensor.

FEBS J 2010 Nov 30;277(22):4651-61. Epub 2010 Sep 30.

Biochemistry Department, University College Cork, Cork, Ireland.

Research on cell-penetrating peptides for the intracellular delivery of porphyrin compounds has mainly focused on the use of trans-activator of transcription (TAT)-derived peptides and, to a lesser extent, on proline-rich peptides and phosphorescent metalloporphyrins. In this article, we describe a novel phosphorescent oxygen-sensitive probe for intracellular use which comprises a bactenecin 7 peptide fragment (15-24) conjugated with the uncharged monofunctional derivative of Pt(II) coproporphyrin I (PEPP0). This probe provides efficient loading of various mammalian cells, including PC12, HCT116, SH-SY5Y and HeLa, via cell-type-dependent uptake mechanisms. The conjugate displays a similar distribution in cytoplasm and mitochondria which allows local oxygen levels to be monitored. Respiratory responses of PC12 cells loaded with the conjugate, measured on a time-resolved fluorescent reader, showed significant cell deoxygenation in response to uncoupling by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone and external hypoxia. Treatment with mitochondrial inhibitors led to a decrease in cell deoxygenation. Although the biophysical properties of this conjugate are similar to those of the phosphorescent intracellular oxygen-sensitive probes described previously, it possesses a number of advantages, including ease of synthesis, high loading efficiency and reliability in physiological experiments with cells. This intracellular probe can be employed for the measurement of intracellular O(2) levels in samples containing mammalian cells using the phosphorescence quenching technique. In addition, the responses to metabolic stimuli can be assessed in a wide range of cells, as can the levels of relative cell oxygenation under external hypoxia.
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http://dx.doi.org/10.1111/j.1742-4658.2010.07872.xDOI Listing
November 2010

Bafilomycin A1 activates respiration of neuronal cells via uncoupling associated with flickering depolarization of mitochondria.

Cell Mol Life Sci 2011 Mar 6;68(5):903-17. Epub 2010 Sep 6.

Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Republic of Ireland.

Bafilomycin A1 (Baf) induces an elevation of cytosolic Ca(2+) and acidification in neuronal cells via inhibition of the V-ATPase. Also, Baf uncouples mitochondria in differentiated PC12 ((d)PC12), (d)SH-SY5Y cells and cerebellar granule neurons, and markedly elevates their respiration. This respiratory response in (d)PC12 is accompanied by morphological changes in the mitochondria and decreases the mitochondrial pH, Ca(2+) and ΔΨm. The response to Baf is regulated by cytosolic Ca(2+) fluxes from the endoplasmic reticulum. Inhibition of permeability transition pore opening increases the depolarizing effect of Baf on the ΔΨm. Baf induces stochastic flickering of the ΔΨm with a period of 20 ± 10 s. Under conditions of suppressed ATP production by glycolysis, oxidative phosphorylation impaired by Baf does not provide cells with sufficient ATP levels. Cells treated with Baf become more susceptible to excitation with KCl. Such mitochondrial uncoupling may play a role in a number of (patho)physiological conditions induced by Baf.
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http://dx.doi.org/10.1007/s00018-010-0502-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037485PMC
March 2011

Dysregulation of hypoxia pathways in fumarate hydratase-deficient cells is independent of defective mitochondrial metabolism.

Hum Mol Genet 2010 Oct 21;19(19):3844-51. Epub 2010 Jul 21.

Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.

Mutations in the gene encoding the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer in affected individuals. FH-associated neoplasia is characterized by defective mitochondrial function and by upregulation of transcriptional pathways mediated by hypoxia-inducible factor (HIF), although whether and by what means these processes are linked has been disputed. We analysed the HIF pathway in Fh1-/- mouse embryonic fibroblasts (MEFs), in FH-defective neoplastic tissues and in Fh1-/- MEFs re-expressing either wild-type or an extra-mitochondrial restricted form of FH. These experiments demonstrated that upregulation of HIF-1alpha occurs as a direct consequence of FH inactivation. Fh1-/- cells accumulated intracellular fumarate and manifested severe impairment of HIF prolyl but not asparaginyl hydroxylation which was corrected by provision of exogenous 2-oxoglutarate (2-OG). Re-expression of the extra-mitochondrial form of FH in Fh1-/- cells was sufficient to reduce intracellular fumarate and to correct dysregulation of the HIF pathway completely, even in cells that remained profoundly defective in mitochondrial energy metabolism. The findings indicate that upregulation of HIF-1alpha arises from competitive inhibition of the 2-OG-dependent HIF hydroxylases by fumarate and not from disruption of mitochondrial energy metabolism.
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http://dx.doi.org/10.1093/hmg/ddq305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2935862PMC
October 2010