Alexander V. Zhdanov

University College Cork | UCC · iEd Hub and School of Pharmacy

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 wi...

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 s...

Biological applications of phosphorescent probes for sensing molecular oxygen (O 2) 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 modificat...

Optochemical sensors are actively used in cell analysis, however existing systems have limitations with respect to their robustness and analytical performance. We have developed advanced multimodal and multi-parametric solid-state pH sensors for cell analysis based on hydrophobic protonable metal-free porphyrins, such as octaethylporphine (OEP), an...

Using several types of Pt-porphyrin based oxygen sensing materials and conditions, we evaluated comparatively four different detection platforms that perform phosphorescence lifetime (PLT) measurements in the microsecond time domain. The time-resolved fluorescence reader Victor 2, which utilises Rapid Lifetime Determination method and Xe-flash lamp...

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....

Mutations in genes encoding cytochrome c oxidase (COX; mitochondrial complex IV) subunits and assembly factors (e.g., SCO1, SCO2, COA6) are linked to severe metabolic syndromes. Notwithstanding that SCO2 is under transcriptional control of tumour suppressor p53, the role of mitochondrial complex IV dysfunction in cancer metabolism remains obscure....

Cell analysis by optochemical sensing represents large and important niche in life and biomedical sciences. We present advanced multi-modal, multi-analyte sensing platform and dedicated materials for cell analysis based on the substituted phosphorescent Pt(II)- or Pd(II)-porphyrin indicator dyes bearing dual O2 and pH sensing functionality (MePor-S...

Identification of tumour margins during resection of the brain is critical for improving the post-operative outcomes. Current methods of tumour identification use 5-ALA, an exogenous precursor, metabolized to fluorescent PpIX in tumour tissue. Although visible under fluorescent microscope, PpIX is easily photo-bleached and tumour tagging is subject...

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...

Background The role of the gut microbiome in the biotransformation of drugs has recently come under scrutiny. It remains unclear whether the gut microbiome directly influences the extent of drug absorbed after oral administration and thus potentially alters clinical pharmacokinetics. Methods In this study, we evaluated whether changes in the gut m...

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 p...

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...

SCO2 (synthesis of cytochrome c oxidase 2) is essential for the assembly and functioning of mitochondrial cytochrome c oxidase (COX) complex (IV). Mutations in the SCO2 gene have been reported to cause COX deficiency and result in fatal infantile cardioencephalomyopathy. Furthermore, the tumor suppressor protein p53 has been shown to mediate its ef...

Significance In this study, we describe a previously unknown mechanism of de novo protein-coding gene evolution. We show that the POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase, is in fact a dual coding gene. Ribosome profiling, phylogenetic conservation, and reporter construct analyses all demonstrate that POLG mRNA...

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 scann...

Microbial spoilage and foodborne diseases cause significant economic and productivity losses. There is a need for novel approaches and antimicrobial treatments to extend shelf life of products, improve quality and microbial safety, and reduce spoilage and waste, and new assessment methods. Traditional assays for testing the toxicity of antimicrobia...

A set of phosphorescent materials were evaluated using a new macro-imager based on fast time-stamping Tpx3Cam camera with phosphorescent lifetime imaging (PLIM) capabilities. The near-infrared emitting O2-sensitive dye PtBP was embedded in several polymers having different permeability for O2 and applied as solid-state coatings on polyester film su...

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 mRNA initiates almost as efficiently (~60-70%) as an AUG in...

The properties of a novel ultra-fast optical imager, Tpx3Cam, were investigated for macroscopic wide-field phosphorescent lifetime imaging (PLIM) applications. The camera is based on a novel optical sensor and Timepix3 readout chip with a time resolution of 1.6 ns, recording of photon arrival time and time over threshold for each pixel, and readout...

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, metab...

The imaging of real-time fluxes of K+ ions in live cell with high dynamic range (5–150 × 10−3 m) 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. It is found th...

Purpose: Optic nerve cupping in glaucoma is characterized by remodeling of the extracellular matrix (ECM) and fibrosis in the lamina cribrosa (LC). We have previously shown that glaucoma LC cells express raised levels of ECM genes and have elevated intracellular calcium ([Ca2+]i). Raised [Ca2+]i is known to promote proliferation, activation, and c...

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...

In the last decade considerable progress has been achieved in the design of various fluorescent and phosphorescent oxygen sensing probes, their delivery to live animal tissue and subsequent imaging of tissue oxygenation. A significant number of publications reflect the growing interests in O2 imaging technologies. The in vivo O2 imaging platforms,...

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 mitocho...

Mitochondrial membrane potential (DYm) is one of the key drivers of free radical production. Changes in DYm strongly affect free radical turnover in both mitochondria and cytosol. We found that in cytochrome c oxidase deficient HCT116 cells F1Fo ATP synthase (mATPase) is reversed in order to maintain DYm polarisation. For small energy costs, mATPas...

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 wit...

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 protoc...

Molecular oxygen (O2) plays a multitude of important roles in cell and tissue function and (patho)physiology. Real-time quantitative imaging of O2 by phosphorescence quenching method enables detailed mechanistic studies of cell and tissue physiology, responses to hypoxia, drug treatment and other stimuli. We applied high-resolution Phosphorescence...

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: Usi...

Hypoxia and reactive oxygen species (ROS) signalling research is a rapidly growing area spanning life and biomedical sciences. Still many current in vitro models and experimental set ups lack physiological relevance, particularly precise in situ control of cell and tissue O2. Quenched-phosphorescence O2 sensing enables implementation of such contro...

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. O2 and ROS signalling and hypoxia research are rapidly growing areas spanning life and biomedical sciences, but still many current cell and ti...

Oxygen plays pivotal role in aerobic metabolism and regulation of cell and tissue function. Local differences and fluctuations in tissue O2 levels are well documented, however physiological significance of O2 micro-gradients, particularly at sub-cellular level, remains poorly understood. Using cell-penetrating phosphorescent O2 probe Pt-Glc and con...

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 (HIF), which makes oncometabolite mimetics broadly utilised in hypoxia research. We found that dimethyloxalylglycine (DM...

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 t...

HIF1α is well known for its role in induction of metastasis leading to cancer progression.LOXL2 and LOX can induce epithelial‐to‐mesenchymal transition (EMT) of tumor cells and turn them into more aggressive phenotype. Recent findings show that LOX expression can be activated by both hypoxia and reactive oxygen species (ROS) via HIF1 pathway, sugge...

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 meta...

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 2–4 h moderate hypoxia, HIF-α protein levels were dictated by the availability of glutamine and glucose, essential for deep c...

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, adeq...

Monitoring of oxygenation is important for physiological experiments investigating the growth, differentiation and function of individual cells in 3D tissue models. Phosphorescence based O-2 sensing and imaging potentially allow this task; however, current probes do not provide the desired bio-distribution and analytical performance. We present sev...

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...

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 t...

Haeme-responsive gene (HRG)-1 encodes a 16-kDa transmembrane protein that is induced by insulin-like growth factor-1 (IGF-1) and associates with the vacuolar-(H(+)) ATPase (V-ATPase). We previously reported that HRG-1 is essential for V-ATPase activity in endosomal acidification and receptor trafficking. Here, we show that in highly invasive and mi...

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 res...

Availability of O 2 determines the cell metabolic status and physiological responses to stimulation. The multicellular spheroids made of cultured or primary neuronal cells are commonly used to study neural stem cell proliferation, differentiation and pathological states under conditions that resemble their native micro‐environment. The live cell im...

Cell and tissue oxygenation in vivo is maintained within narrow physiological range and its significant deviations from the norm can lead to pathological states. Real‐time monitoring of this parameter and oxygen consumption rate (OCR) in respiring samples provides valuable tool for cell metabolic assessment. We describe two new phosphorescent probe...

Monitoring cell and tissue oxygenation is important for the analysis of cell development and differentiation, mitochondrial function, and common (patho)physiological conditions such as ischemia, cancer, neurodegenerative disorders. A number of materials for sensing cellular oxygen (O2) by optical means have been described in recent years, but the d...

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 bafilomycin A1 (Baf) causes partial depolarization of the mitochondria and deep sustained deoxygenation of human colon cancer HCT11...

This chapter describes the fundamentals of an O2 imaging technique based on the quenched-phosphorescence detection of Pt-porphyrin probes. The wide-field, confocal and multi-photon microscopy and methodological aspects of quenched phosphorescence icO2 imaging techniques, theoretical and practical considerations, are briefly described and critically...

In this chapter, the use of Pt-porphyrin-based extracellular and intracellular O2 sensing probes (ecO2 and icO2) on a TR-F plate reader format is described and critically assessed. The principles underpinning extracellular measurement are outlined and the assessment of biological oxygen consumption in prokaryotic cells, eukaryotic cells and small o...

The role of acetylation of histone H4 tail at residue K16 (H4K16Ac) in epigenetic regulation is widely recognized as the mark of nucleosome de‐compactization and activation of gene expression. The enzymes responsible for this modification are histone acetyl transferases (HATs) which use acetyl‐CoA (ACA) as a substrate. Recently it has been shown th...

The diffusion of oxygen (O 2 ) into tissue, cell and mitochondria regulates metabolism, gene expression and cell fate. Depending on the cell type and mitochondrial function the gradient between extracellular and intracellular O 2 may vary and is hypothesized to play a physiological role, i.e. stabilization of HIF protein. With numerous approaches p...

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 a...

Monitoring cell and tissue oxygenation is important for the analysis of cell development and differentiation, mitochondrial function, and common (patho)physiological conditions such as ischemia, cancer, neurodegenerative disorders. A number of materials for sensing cellular oxygen (O-2) by optical means have been described in recent years, but the...

The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and t...

Cancer cells exhibit increases glycolytic rates and glucose transport to facilitate adaptation to the high energy demand imposed by increased proliferation rates and their often hypoxic environment. Moreover, this is often accompanied by reduced mitochondrial activity due to the accumulation of mtDNA mutations. Since there is close coordination of...

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, parti...