Monitoring and modelling the glutamine metabolic pathway: a review and future perspectives.

BACKGROUND: Analysis of the glutamine metabolic pathway has taken a special place in metabolomics research in recent years, given its important role in cell biosynthesis and bioenergetics across several disorders, especially in cancer cell survival. The science of metabolomics addresses the intricate intracellular metabolic network by exploring and understanding how cells function and respond to external or internal perturbations to identify potential therapeutic targets. However, despite recent advances in metabolomics, monitoring the kinetics of a metabolic pathway in a living cell in situ, real-time and holistically remains a significant challenge. AIM: This review paper explores the range of analytical approaches for monitoring metabolic pathways, as well as physicochemical modeling techniques, with a focus on glutamine metabolism. We discuss the advantages and disadvantages of each method and explore the potential of label-free Raman microspectroscopy, in conjunction with kinetic modeling, to enable real-time and in situ monitoring of the cellular kinetics of the glutamine metabolic pathway. KEY SCIENTIFIC CONCEPTS: Given its important role in cell metabolism, the ability to monitor and model the glutamine metabolic pathways are highlighted. Novel, label free approaches have the potential to revolutionise metabolic biosensing, laying the foundation for a new paradigm in metabolomics research and addressing the challenges in monitoring metabolic pathways in living cells.

Antibacterial activity of metal-phenanthroline complexes against multidrug-resistant Irish clinical isolates: a whole genome sequencing approach.

Antimicrobial resistance (AMR) is one of the serious global health challenges of our time. There is now an urgent need to develop novel therapeutic agents that can overcome AMR, preferably through alternative mechanistic pathways from conventional treatments. The antibacterial activity of metal complexes (metal = Cu(II), Mn(II), and Ag(I)) incorporating 1,10-phenanthroline (phen) and various dianionic dicarboxylate ligands, along with their simple metal salt and dicarboxylic acid precursors, against common AMR pathogens were investigated. Overall, the highest level of antibacterial activity was evident in compounds that incorporate the phen ligand compared to the activities of their simple salt and dicarboxylic acid precursors. The chelates incorporating both phen and the dianion of 3,6,9-trioxaundecanedioic acid (tdda) were the most effective, and the activity varied depending on the metal centre. Whole-genome sequencing (WGS) was carried out on the reference Pseudomonas aeruginosa strain, PAO1. This strain was exposed to sub-lethal doses of lead metal-tdda-phen complexes to form mutants with induced resistance properties with the aim of elucidating their mechanism of action. Various mutations were detected in the mutant P. aeruginosa genome, causing amino acid changes to proteins involved in cellular respiration, the polyamine biosynthetic pathway, and virulence mechanisms. This study provides insights into acquired resistance mechanisms of pathogenic organisms exposed to Cu(II), Mn(II), and Ag(I) complexes incorporating phen with tdda and warrants further development of these potential complexes as alternative clinical therapeutic drugs to treat AMR infections.

The Antibacterial Drug Candidate SBC3 is a Potent Inhibitor of Bacterial Thioredoxin Reductase.

Antibiotic resistance is a growing problem for public health and associated with increasing economic costs and mortality rates. Silver and silver-related compounds have been used for centuries due to their antimicrobial properties. In this work, we show that 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene silver(I) acetate/NHC*-Ag-OAc (SBC3) is a reversible, high affinity inhibitor of E. coli thioredoxin reductase (TrxR; Ki =10.8±1.2 nM). Minimal inhibition concentration (MIC) tests with different E. coli and P. aeruginosa strains demonstrated that SBC3 can efficiently inhibit bacterial cell growth, especially in combination with established antibiotics like gentamicin. Our results show that SBC3 is a promising antibiotic drug candidate targeting bacterial TrxR.

A 4-Gene Signature of CDKN1, FDXR, SESN1 and PCNA Radiation Biomarkers for Prediction of Patient Radiosensitivity.

The quest for the discovery and validation of radiosensitivity biomarkers is ongoing and while conventional bioassays are well established as biomarkers, molecular advances have unveiled new emerging biomarkers. Herein, we present the validation of a new 4-gene signature panel of CDKN1, FDXR, SESN1 and PCNA previously reported to be radiation-responsive genes, using the conventional G2 chromosomal radiosensitivity assay. Radiation-induced G2 chromosomal radiosensitivity at 0.05 Gy and 0.5 Gy IR is presented for a healthy control (n = 45) and a prostate cancer (n = 14) donor cohort. For the prostate cancer cohort, data from two sampling time points (baseline and Androgen Deprivation Therapy (ADT)) is provided, and a significant difference (p > 0.001) between 0.05 Gy and 0.5 Gy was evident for all donor cohorts. Selected donor samples from each cohort also exposed to 0.05 Gy and 0.5 Gy IR were analysed for relative gene expression of the 4-gene signature. In the healthy donor cohort, there was a significant difference in gene expression between IR dose for CDKN1, FXDR and SESN1 but not PCNA and no significant difference found between all prostate cancer donors, unless they were classified as radiation-induced G2 chromosomal radiosensitive. Interestingly, ADT had an effect on radiation response for some donors highlighting intra-individual heterogeneity of prostate cancer donors.

Non-thermal atmospheric plasma induces ROS-independent cell death in U373MG glioma cells and augments the cytotoxicity of temozolomide.

BACKGROUND: Non-thermal atmospheric plasma (NTAP) is an ionised gas produced under high voltage that can generate short-lived chemically active species and induce a cytotoxic insult in cancer cells. Cell-specific resistance to NTAP-mediated cytotoxicity has been reported in the literature. The aim of this study was to determine whether resistance against NTAP could be overcome using the human glioma cell line U373MG. METHODS: Non-thermal atmospheric plasma was generated using a Dielectric Barrier Device (DBD) system with a maximum voltage output of 120 kV at 50 Hz. The viability of U373MG GBM cells and HeLa cervical carcinoma cells was determined using morphology, flow cytometry and cytotoxicity assays. Fluorescent probes and inhibitors were used to determine the mechanisms of cytotoxicity of cells exposed to the plasma field. Combinational therapy with temozolomide (TMZ) and multi-dose treatments were explored as mechanisms to overcome resistance to NTAP. RESULTS: Non-thermal atmospheric plasma decreased cell viability in a dose (time)-dependent manner. U373MG cells were shown to be resistant to NTAP treatment when compared with HeLa cells, and the levels of intracellular reactive oxygen species (ROS) quantified in U373MG cells were much lower than in HeLa cells following exposure to the plasma field. Reactive oxygen species inhibitor N-acetyl cysteine (NAC) only alleviated the cytotoxic effects in HeLa cells and not in the relatively NTAP-resistant cell line U373MG. Longer exposures to NTAP induced a cell death independent of ROS, JNK and caspases in U373MG. The relative resistance of U373MG cells to NTAP could be overcome when used in combination with low concentrations of the GBM chemotherapy TMZ or exposure to multiple doses. CONCLUSIONS: For the very first time, we report that NTAP induces an ROS-, JNK- and caspase-independent mechanism of cell death in the U373MG GBM cell line that can be greatly enhanced when used in combination with low doses of TMZ. Further refinement of the technology may facilitate localised activation of cytotoxicity against GBM when used in combination with new and existing chemotherapeutic regimens.

Vibrational spectroscopy in sensing radiobiological effects: analyses of targeted and non-targeted effects in human keratinocytes.

Modern models of radiobiological effects include mechanisms of damage initiation, sensing and repair, for those cells that directly absorb ionizing radiation as well as those that experience molecular signals from directly irradiated cells. In the former case, the effects are termed targeted effects while, in the latter, non-targeted effects. It has emerged that phenomena occur at low doses below 1 Gy in directly irradiated cells that are associated with cell-cycle-dependent mechanisms of DNA damage sensing and repair. Likewise in non-targeted bystander-irradiated cells the effect saturates at 0.5 Gy. Both effects at these doses challenge the limits of detection of vibrational spectroscopy. In this paper, a study of the sensing of both targeted and non-targeted effects in HaCaT human keratinocytes irradiated with gamma ray photons is conducted with vibrational spectroscopy. In the case of directly irradiated cells, it is shown that the HaCaT cell line does exhibit both hyperradiosensitivity and increased radioresistance at low doses, a transition between the two effects occurring at a dose of 200 mGy, and that cell survival and other physiological effects as a function of dose follow the induced repair model. Both Raman and FTIR signatures are shown to follow a similar model, suggesting that the spectra include signatures of DNA damage sensing and repair. In bystander-irradiated cells, pro- and anti-apoptotic signalling and mechanisms of ROS damage were inhibited in the mitogen-activated protein kinase (MAPK) transduction pathway. It is shown that Raman spectral profiles of bystander-irradiated cells are correlated with markers of bystander signalling and molecular transduction. This work demonstrates for the first time that both targeted and non-targeted effects of ionizing radiation damage are detected by vibrational spectroscopy in vitro.

Apoptosis is signalled early by low doses of ionising radiation in a radiation-induced bystander effect.

It is known that ionising radiation (IR) induces a complex signalling apoptotic cascade post-exposure to low doses ultimately to remove damaged cells from a population, specifically via the intrinsic pathway. Therefore, it was hypothesised that bystander reporter cells may initiate a similar apoptotic response if exposed to low doses of IR (0.05Gy and 0.5Gy) and compared to directly irradiated cells. Key apoptotic genes were selected according to their role in the apoptotic cascade; tumour suppressor gene TP53, pro-apoptotic Bax and anti-apoptotic Bcl2, pro-apoptotic JNK and anti-apoptotic ERK, initiator caspase 2 and 9 and effector caspase 3, 6 and 7. The data generated consolidated the role of apoptosis following direct IR exposure for all doses and time points as pro-apoptotic genes such as Bax and JNK as well as initiator caspase 7 and effector caspase 3 and 9 were up-regulated. However, the gene expression profile for the bystander response was quite different and more complex in comparison to the direct response. The 0.05Gy dose point had a more significant apoptosis gene expression profile compared to the 0.5Gy dose point and genes were not always expressed within 1h but were sometimes expressed 24h later. The bystander data clearly demonstrates initiation of the apoptotic cascade by the up-regulation of TP53, Bax, Bcl-2, initiator caspase 2 and effector caspase 6. The effector caspases 3 and 7 of the bystander samples demonstrated down-regulation in their gene expression levels at 0.05Gy and 0.5Gy at both time points therefore not fully executing the apoptotic pathway. Extensive analysis of the mean-fold gene expression changes of bystander data demonstrated that the apoptosis is initiated in the up-regulation of pro-apoptotic and initiator genes but may not very well be executed to final stages of cell death due to down-regulation of effector genes.

Structural and solution speciation studies on selected [Cu(NN)(OO)] complexes and an investigation of their biomimetic activity, ROS generation and their cytotoxicity in normoxic, hypoxic and anoxic environments in MCF-7 breast cancer-derived cells.

Reactive oxygen species(ROS) generation with subsequent DNA damage is one of the principle mechanisms of action assigned to copper-based anticancer complexes. The efficacy of this type of chemotherapeutic may be reduced in the low oxygen environment of tumours. In this study the cytotoxicity of three complexes, [Cu(dips)(phen)] (1), [Cu(ph)(phen)]·2H2O (2) and [Cu(ph)(bpy)]·H2O (3) (disp: 3,5-diisopropylsalicylate, phen: 1,10- phenanthroline, ph: phthalate, bpy: 2,2'-bipyridyl) were assessed for anticancer activity in the breast-cancer derived MCF-7 line under normoxic, hypoxic and anoxic conditions. In an immortalised keratinocyte HaCaT cell line, the cytotoxicity of complexes 2 and 3 was significantly reduced under both normoxic and hypoxic conditions, whilst the cytotoxicity of complex 1 was increased under hypoxic conditions. The ability of the complexes to generate ROS in the MCF-7 cell line was evaluated as was their ability to act as superoxide dismutase(SOD) and catalase mimics using a yeast cell assay. ROS generation was significant for complexes 2 and 3, less so for complex 1 though all three complexes had SOD mimetic ability. Given the ternary nature of the complexes, solution speciation studies were undertaken but were only successful for complex 3, due to solubility issues with the other two complexes. The concentration distribution of various species, formed in aqueous solution, was evaluated as a function of pH and confirmed that complex 3 is the dominant species at physiological pH in the mM concentration range. However, as its concentration diminishes, it experiences a progressive dissociation, leading to the formation of binary complexes of bpy alongside unbound phthalate.

Quantitative reagent-free detection of fibrinogen levels in human blood plasma using Raman spectroscopy.

Fibrinogen assays are commonly used as part of clinical screening tests to investigate haemorrhagic states, for detection of disseminated intravascular coagulation and as a predictor of a variety of cardiovascular events. The Clauss assay, which measures thrombin clotting time, is the most commonly used method for measuring fibrinogen levels. Nevertheless, inconsistencies are present in inter-manufacturer reagent sources, calibration standards and methodologies. Automated coagulation analysers, which measure changes in optical density during the prothrombin time (PT-Fg), have found use in many hospitals. However, the PT-Fg method is found to give falsely elevated values due to varying choices of calibrants, reagents and analysers. As an alternative, Raman spectroscopy has previously been applied to the analysis of blood and its various constituents to determine various analyte concentrations such as glucose, urea, triglycerides and cholesterol. In this study, Raman spectroscopy was investigated for its ability to accurately quantify fibrinogen concentration in blood plasma. Samples collected from 34 patients were analysed by Raman spectroscopy and the resultant spectra were fitted with a Partial Least Squares Regression model using target values obtained through a pre-calibrated Clauss fibrinogen assay. Various spectral pre-processing methods were utilised to prepare data to be entered into a calibration model. A root mean square error of prediction of 0.72 ± 0.05 g/L was achieved with as few as 25 spectra. In this pilot study, Raman spectroscopy has been demonstrated to be a robust technique providing rapid and reagent-free quantification of fibrinogen levels in blood plasma and a potential alternative to the Clauss assay.

Monitoring and modelling the dynamics of the cellular glycolysis pathway: A review and future perspectives.

BACKGROUND: The dynamics of the cellular glycolysis pathway underpin cellular function and dysfunction, and therefore ultimately health, disease, diagnostic and therapeutic strategies. Evolving our understanding of this fundamental process and its dynamics remains critical. SCOPE OF REVIEW: This paper reviews the medical relevance of glycolytic pathway in depth and explores the current state of the art for monitoring and modelling the dynamics of the process. The future perspectives of label free, vibrational microspectroscopic techniques to overcome the limitations of the current approaches are considered. MAJOR CONCLUSIONS: Vibrational microspectroscopic techniques can potentially operate in the niche area of limitations of other omics technologies for non-destructive, real-time, in vivo label-free monitoring of glycolysis dynamics at a cellular and subcellular level.

Women's contributions to radiobiology in Ireland; from small beginnings .

OBJECTIVES: To describe the contribution of women radiobiologists in Ireland to the development of the discipline internationally and at home and to discuss the history of radiobiology in Ireland to date. This parallels the history of the evolution of a small radiobiology group in Kevin Street, Dublin Institute of Technology (DIT) which was formerly part of the City of Dublin Vocational Education Committee. There followed years of development first as a radiobiological research center which evolved in the FOCAS Research Institute now embedded within Technological University Dublin (TU Dublin). CONCLUSIONS: Over the last 45 years, the women of the Radiation and Environmental Science Centre (RESC) contributed to the major paradigm shift in low dose radiobiology contributing exciting new research concerning non-targeted effects, including discovery of lethal mutations, medium transfer bystander mechanisms, and signaling pathways. They also developed translational research using human explant culture systems with unique immunocytochemical methods and more recently evolved to molecular and spectroscopic analysis of clinical samples. The RESC also developed unique in vitro research methods into effects of radiation on non-human species of concern in ecosystems.

In Vivo Activity of Metal Complexes Containing 1,10-Phenanthroline and 3,6,9-Trioxaundecanedioate Ligands against Pseudomonas aeruginosa Infection in Galleria mellonella Larvae.

Drug-resistant Pseudomonas aeruginosa is rapidly developing resulting in a serious global threat. Immunocompromised patients are specifically at risk, especially those with cystic fibrosis (CF). Novel metal complexes incorporating 1,10-phenanthroline (phen) ligands have previously demonstrated antibacterial and anti-biofilm effects against resistant P. aeruginosa from CF patients in vitro. Herein, we present the in vivo efficacy of {[Cu(3,6,9-tdda)(phen)2]·3H2O·EtOH}n (Cu-tdda-phen), {[Mn(3,6,9-tdda)(phen)2]·3H2O·EtOH}n (Mn-tdda-phen) and [Ag2(3,6,9-tdda)(phen)4]·EtOH (Ag-tdda-phen) (tddaH2 = 3,6,9-trioxaundecanedioic acid). Individual treatments of these metal-tdda-phen complexes and in combination with the established antibiotic gentamicin were evaluated in vivo in larvae of Galleria mellonella infected with clinical isolates and laboratory strains of P. aeruginosa. G. mellonella were able to tolerate all test complexes up to 10 µg/larva. In addition, the immune response was affected by stimulation of immune cells (hemocytes) and genes that encode for immune-related peptides, specifically transferrin and inducible metallo-proteinase inhibitor. The amalgamation of metal-tdda-phen complexes and gentamicin further intensified this response at lower concentrations, clearing a P. aeruginosa infection that were previously resistant to gentamicin alone. Therefore this work highlights the anti-pseudomonal capabilities of metal-tdda-phen complexes alone and combined with gentamicin in an in vivo model.

Ursolic Acid Inhibits Collective Cell Migration and Promotes JNK-Dependent Lysosomal Associated Cell Death in Glioblastoma Multiforme Cells.

Ursolic acid (UA) is a bioactive compound which has demonstrated therapeutic efficacy in a variety of cancer cell lines. UA activates various signalling pathways in Glioblastoma multiforme (GBM) and offers a promising starting point in drug discovery; however, understanding the relationship between cell death and migration has yet to be elucidated. UA induces a dose dependent cytotoxic response demonstrated by flow cytometry and biochemical cytotoxicity assays. Inhibitor and fluorescent probe studies demonstrate that UA induces a caspase independent, JNK dependent, mechanism of cell death. Migration studies established that UA inhibits GBM collective cell migration in a time dependent manner that is independent of the JNK signalling pathway. Cytotoxicity induced by UA results in the formation of acidic vesicle organelles (AVOs), speculating the activation of autophagy. However, inhibitor and spectrophotometric analysis demonstrated that autophagy was not responsible for the formation of the AVOs. Confocal microscopy and isosurface visualisation determined co-localisation of lysosomes with the previously identified AVOs, thus providing evidence that lysosomes are likely to be playing a role in UA induced cell death. Collectively, our data identify that UA rapidly induces a lysosomal associated mechanism of cell death in addition to UA acting as an inhibitor of GBM collective cell migration.

Evaluation of the potential of Raman microspectroscopy for prediction of chemotherapeutic response to cisplatin in lung adenocarcinoma.

The study of the interaction of anticancer drugs with mammalian cells in vitro is important to elucidate the mechanisms of action of the drug on its biological targets. In this context, Raman spectroscopy is a potential candidate for high throughput, non-invasive analysis. To explore this potential, the interaction of cis-diamminedichloroplatinum(II) (cisplatin) with a human lung adenocarcinoma cell line (A549) was investigated using Raman microspectroscopy. The results were correlated with parallel measurements from the MTT cytotoxicity assay, which yielded an IC(50) value of 1.2 ± 0.2 µM. To further confirm the spectral results, Raman spectra were also acquired from DNA extracted from A549 cells exposed to cisplatin and from unexposed controls. Partial least squares (PLS) multivariate regression and PLS Jackknifing were employed to highlight spectral regions which varied in a statistically significant manner with exposure to cisplatin and with the resultant changes in cellular physiology measured by the MTT assay. The results demonstrate the potential of the cellular Raman spectrum to non-invasively elucidate spectral changes that have their origin either in the biochemical interaction of external agents with the cell or its physiological response, allowing the prediction of the cellular response and the identification of the origin of the chemotherapeutic response at a molecular level in the cell.

The Antibacterial and Anti-biofilm Activity of Metal Complexes Incorporating 3,6,9-Trioxaundecanedioate and 1,10-Phenanthroline Ligands in Clinical Isolates of Pseudomonas Aeruginosa from Irish Cystic Fibrosis Patients.

Chronic infections of Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients are problematic in Ireland where inherited CF is prevalent. The bacteria's capacity to form a biofilm in its pathogenesis is highly virulent and leads to decreased susceptibility to most antibiotic treatments. Herein, we present the activity profiles of the Cu(II), Mn(II) and Ag(I) tdda-phen chelate complexes {[Cu(3,6,9-tdda)(phen)2].3H2O.EtOH}n (Cu-tdda-phen), {[Mn(3,6,9-tdda)(phen)2].3H2O.EtOH}n (Mn-tdda-phen) and [Ag2(3,6,9-tdda)(phen)4].EtOH (Ag-tdda-phen) (tddaH2 = 3,6,9-trioxaundecanedioic acid; phen = 1,10-phenanthroline) towards clinical isolates of P. aeruginosa derived from Irish CF patients in comparison to two reference laboratory strains (ATCC 27853 and PAO1). The effects of the metal-tdda-phen complexes and gentamicin on planktonic growth, biofilm formation (pre-treatment) and mature biofilm (post-treatment) alone and in combination were investigated. The effects of the metal-tdda-phen complexes on the individual biofilm components; exopolysaccharide, extracellular DNA (eDNA), pyocyanin and pyoverdine are also presented. All three metal-tdda-phen complexes showed comparable and often superior activity to gentamicin in the CF strains, compared to their activities in the laboratory strains, with respect to both biofilm formation and established biofilms. Combination studies presented synergistic activity between all three complexes and gentamicin, particularly for the post-treatment of established mature biofilms, and was supported by the reduction of the individual biofilm components examined.

Pt(IV) pro-drugs with an axial HDAC inhibitor demonstrate multimodal mechanisms involving DNA damage and apoptosis independent of cisplatin resistance in A2780/A2780cis cells.

Epigenetic agents such as histone deacetylase (HDAC) inhibitors are widely investigated for use in combined anticancer therapy and the co-administration of Pt drugs with HDAC inhibitors has shown promise for the treatment of resistant cancers. Coordination of an HDAC inhibitor to an axial position of a Pt(IV) derivative of cisplatin allows the combination of the epigenetic drug and the Pt chemotherapeutic into a single molecule. In this work we carry out mechanistic studies on the known Pt(IV) complex cis,cis,trans-[Pt(NH3)2Cl2(PBA)2] (B) with the HDAC inhibitor 4-phenylbutyrate (PBA) and its derivatives cis,cis,trans-[Pt(NH3)2Cl2(PBA)(OH)] (A), cis,cis,trans-[Pt(NH3)2Cl2(PBA)(Bz)] (C), and cis,cis,trans-[Pt(NH3)2Cl2(PBA)(Suc)] (D) (Bz = benzoate, Suc = succinate). The comparison of the cytotoxicity, effect on HDAC activity, reactive oxygen species (ROS) generation, γ-H2AX (histone 2A-family member X) foci generation and induction of apoptosis in cisplatin-sensitive and cisplatin-resistant ovarian cancer cells shows that A - C exhibit multimodal mechanisms involving DNA damage and apoptosis independent of cisplatin resistance.

Cu(ii) phenanthroline-phenazine complexes dysregulate mitochondrial function and stimulate apoptosis.

Herein we report an in-depth study on the cytotoxic mechanism of action of four developmental cytotoxic copper(ii) complexes: [Cu(phen)2]2+ (Cu-Phen); [Cu(DPQ)(Phen)]2+ (Cu-DPQ-Phen); [Cu(DPPZ)(Phen)]2+; and [Cu(DPPN)(Phen)]2+ (where Phen = 1,10-phenanthroline, DPQ = dipyrido[3,2-f:2',3'-h]quinoxaline, DPPZ = dipyrido[3,2-a:2',3'-c]phenazine, and DPPN = benzo[i]dipyrido[3,2-a:2',3'-c]phenazine). This complex class is known for its DNA intercalative properties and recent evidence-derived from an in vivo proteomic study-supports the potential targeting of mitochondrial function. Therefore, we focused on mitochondrial-mediated apoptosis related to cytotoxic activity and the potential impact these agents have on mitochondrial function. The Cu(ii) complexes demonstrated superior activity regardless of aromatic extension within the phenazine ligand to the previously demonstrated activity of cisplatin. Unique toxicity mechanisms were also identified in prior demonstrated cisplatin sensitive and resistant cell lines. Double strand breaks in genomic DNA, quantified by γH2AX foci formation, were then coupled with apoptotic gene expression to elucidate the mechanisms of cell death. These results indicate that while DNA damage-induced apoptosis by BAX, XIAP and caspase-9 and -3 expression is moderate for the Cu(ii) complexes when compared to cisplatin, protein targets independent of DNA exert a multimodal mechanistic effect. Significantly, mitochondrial gene expression of oxidative stress, protease expression, and fission/fusion processes-upregulated HMOX, DRP1 and LON, respectively-indicated an increased oxidative damage associated with compromised mitochondrial health upon exposure to these agents. These data support a unique mode of action by these complexes and provide valuable evidence of the developmental potential of these therapeutic inorganic complexes.

MicroRNA Analysis of ATM-Deficient Cells Indicate PTEN and CCDN1 as Potential Biomarkers of Radiation Response.

Genetic and epigenetic profile changes associated with individual radiation sensitivity are well documented and have led to enhanced understanding of the mechanisms of the radiation-induced DNA damage response. However, the search continues to identify reliable biomarkers of individual radiation sensitivity. Herein, we report on a multi-biomarker approach using traditional cytogenetic biomarkers, DNA damage biomarkers and transcriptional microRNA (miR) biomarkers coupled with their potential gene targets to identify radiosensitivity in ataxia-telangiectasia mutated (ATM)-deficient lymphoblastoid cell lines (LCL); ATM-proficient cell lines were used as controls. Cells were 0.05 and 0.5 Gy irradiated, using a linear accelerator, with sham-irradiated cells as controls. At 1 h postirradiation, cells were fixed for γ-H2AX analysis as a measurement of DNA damage, and cytogenetic analysis using the G2 chromosomal sensitivity assay, G-banding and FISH techniques. RNA was also isolated for genetic profiling by microRNA (miR) and RT-PCR analysis. A panel of 752 miR were analyzed, and potential target genes, phosphatase and tensin homolog (PTEN) and cyclin D1 (CCND1), were measured. The cytogenetic assays revealed that although the control cell line had functional cell cycle checkpoints, the radiosensitivity of the control and AT cell lines were similar. Analysis of DNA damage in all cell lines, including an additional control cell line, showed elevated γ-H2AX levels for only one AT cell line. Of the 752 miR analyzed, eight miR were upregulated, and six miR were downregulated in the AT cells compared to the control. Upregulated miR-152-3p, miR-24-5p and miR-92-15p and all downregulated miR were indicated as modulators of PTEN and CCDN1. Further measurement of both genes validated their potential role as radiation-response biomarkers. The multi-biomarker approach not only revealed potential candidates for radiation response, but provided additional mechanistic insights into the response in AT-deficient cells.

In vivo Activity of Copper(II), Manganese(II), and Silver(I) 1,10-Phenanthroline Chelates Against Candida haemulonii Using the Galleria mellonella Model.

Candida haemulonii is an emerging opportunistic pathogen resistant to most antifungal drugs currently used in clinical arena. Metal complexes containing 1,10-phenanthroline (phen) chelating ligands have well-established anti-Candida activity against different medically relevant species. This study utilized larvae of Galleria mellonella, a widely used model of in vivo infection, to examine C. haemulonii infection characteristics in response to different copper(II), manganese(II), and silver(I) chelates containing phen, which had demonstrated potent anti-C. haemulonii activity in a previous study. The results showed that C. haemulonii virulence was influenced by inoculum size and incubation temperature, and the host G. mellonella immune response was triggered in an inoculum-dependent manner reflected by the number of circulating immune cells (hemocytes) and observance of larval melanization process. All test chelates were non-toxic to the host in concentrations up to 10 µg/larva. The complexes also affected the G. mellonella immune system, affecting the hemocyte number and the expression of genes encoding antifungal and immune-related peptides (e.g., inducible metalloproteinase inhibitor protein, transferrin, galiomycin, and gallerimycin). Except for [Ag2(3,6,9-tdda)(phen)4].EtOH (3,6,9-tddaH2 = 3,6,9-trioxoundecanedioic acid), all chelates were capable of affecting the fungal burden of infected larvae and the virulence of C. haemulonii in a dose-dependent manner. This work shows that copper(II), manganese(II), and silver(I) chelates containing phen with anti-C. haemulonii activity are capable of (i) inhibiting fungal proliferation during in vivo infection, (ii) priming an immune response in the G. mellonella host and (iii) affecting C. haemulonii virulence.

Do radiation-induced bystander effects correlate to the intrinsic radiosensitivity of individuals and have clinical significance?

It is well known that patients can vary in their normal tissue response to radiotherapy, and this can be problematic. As a result, radiobiologists have been using in vitro models to assess variation in response and elucidate the genetic determinants of this variation. However, the clinical relevance of these models is currently unknown. In this study, blood samples from healthy controls (n = 20) and colorectal carcinoma patients (n = 60) were cultured in vitro to assess two radiobiological end points in parallel: intrinsic radiosensitivity assayed by chromosomal aberrations (G(2) scores) and radiation-induced bystander effects assayed by viability testing. Increased intrinsic radiosensitivity was observed in colorectal carcinoma donors (55%) compared to the healthy donors (5%) (P < 0.005). Similarly, more pronounced radiation-induced bystander effects were observed in the colorectal carcinoma donors compared to the healthy donors after 24 h exposure but not after 96 h exposure to donor irradiated cell conditioned medium (ICCM) (P < 0.05). All scores were tested for correlation with the age, sex and clinical stage of the colorectal carcinoma patients. The only statistically significant correlation was found in samples from severe Dukes D patients (P < 0.005), which had low/radioresistant G(2) scores. No correlation was found between radiation-induced intrinsic sensitivity and bystander effects, which suggests that they may have separate underlying molecular mechanisms, but they both show clinical relevance in individual patient samples.