Brief inactivation of c-Myc is not sufficient for sustained regression of c-Myc-induced tumours of pancreatic islets and skin epidermis

Background Tumour regression observed in many conditional mouse models following oncogene inactivation provides the impetus to develop, and a platform to preclinically evaluate, novel therapeutics to inactivate specific oncogenes. Inactivating single oncogenes, such as c-Myc, can reverse even advanced tumours. Intriguingly, transient c-Myc inactivation proved sufficient for sustained osteosarcoma regression; the resulting osteocyte differentiation potentially explaining loss of c-Myc's oncogenic properties. But would this apply to other tumours? Results We show that brief inactivation of c-Myc does not sustain tumour regression in two distinct tissue types; tumour cells in pancreatic islets and skin epidermis continue to avoid apoptosis after c-Myc reactivation, by virtue of Bcl-xL over-expression or a favourable microenvironment, respectively. Moreover, tumours progress despite reacquiring a differentiated phenotype and partial loss of vasculature during c-Myc inactivation. Interestingly, reactivating c-Myc in β-cell tumours appears to result not only in further growth of the tumour, but also re-expansion of the accompanying angiogenesis and more pronounced β-cell invasion (adenocarcinoma). Conclusions Given that transient c-Myc inactivation could under some circumstances produce sustained tumour regression, the possible application of this potentially less toxic strategy in treating other tumours has been suggested. We show that brief inactivation of c-Myc fails to sustain tumour regression in two distinct models of tumourigenesis: pancreatic islets and skin epidermis. These findings challenge the potential for cancer therapies aimed at transient oncogene inactivation, at least under those circumstances where tumour cell differentiation and alteration of epigenetic context fail to reinstate apoptosis. Together, these results suggest that treatment schedules will need to be informed by knowledge of the molecular basis and environmental context of any given cancer

Ανάπτυξη μεθόδων προσδιορισμού κυανοτοξινών και οργανικών ρύπων σε νερά με υγροχρωματογραφία-συζευγμένη φασματομετρία μαζών

Τα κυανοβακτήρια αναπτύσσονται στο υδάτινο περιβάλλον και κάτω από ευνοϊκές συνθήκες πολλαπλασιάζονται σχηματίζοντας έντονες ανθίσεις (blooms) ελευθερώνοντας συχνά στο νερό τοξικούς δευτερογενείς μεταβολίτες, τις κυανοτοξίνες που αποτελούν σημαντικό κίνδυνο για τα υδάτινα οικοσυστήματα, τους ταμιευτήρες πόσιμου νερού και την δημόσια υγεία. Οι κυανοτοξίνες είναι μία μεγάλη ομάδα ενώσεων με ποικίλες φυσικοχημικές ιδιότητες, χημικές δομές και τοξική δράση (ηπατοτοξικές, νευροτοξικές, δερματοτοξικές, κυτταροτοξικές). Οι κυανοτοξίνες κατηγοριοποιούνται κυρίως με βάση τη δομή τους σε κυκλικά πεπτίδια (όπως Microcystins, Nodularins), αλκαλοειδή (όπως Cylindrospermopsin, Anatoxin), λιποπολυσακχαρίτες και αμινοξέα. Οι περισσότερες μελέτες για την ανάλυση των κυανοτοξινών εστιάζουν στον προσδιορισμό μίας κατηγορίας κυανοτοξινών. Στην παρούσα εργασία αναπτύχθηκε μία γρήγορη, απλή και ευαίσθητη αναλυτική μέθοδος για τον ταυτόχρονο προσδιορισμό διαφορετικών κατηγοριών κυανοτοξινών (Cylindrospermopsin, Anatoxin-a, Nodularin, 12 Microcystins ([D-Asp3]MC-RR, MC-RR, MC-YR, MC-HtyR,[D-Asp3]MC-LR, MC-LR, MC-HilR, MC-WR, MC-LA, MC-LY, MC-LW και MC-LF), καθώς και των πολύ διαδεδομένων θαλάσσιων φυκοτοξινών Okadaic acid και Domoic acid στο νερό με μία μόνο ανάλυση. Ο προσδιορισμός των ενώσεων έγινε με υγροχρωματογραφία – συζευγμένη φασματομετρία μαζών (LC-MS/MS). Για την προκατεργασία του δείγματος και την αποτελεσματική εκχύλιση όλων των αναλυτών από το νερό αναπτύχθηκε μέθοδος εκχύλισης στερεάς φάσης (SPE), με διάταξη διπλού φυσιγγίου αποτελούμενη από Oasis HLB και Hyper PGC. Επιπλέον, αναπτύχθηκε μέθοδος για την ταυτόχρονη εκχύλιση διαφορετικών κατηγοριών κυανοτοξινών (Cylindrospermopsin, Anatoxin-a and Microcystins) από λυοφιλοποιημένη κυανοβακτηριακή βιομάζα, όπου μελετήθηκε η επίδραση διάφορων παραγόντων στην απόδοση της εκχύλισης. Οι αναπτυχθείσες μέθοδοι εφαρμόστηκαν αποτελεσματικά στην ανάλυση δειγμάτων από λίμνες της Ελλάδας, όπου ανιχνεύθηκε και ταυτοποιήθηκε για πρώτη φορά στον ελλαδικό χώρο η παρουσία των κυανοτοξινών Cylindrospermopsin, Anatoxin-a και ποικίλων Microcystins. Οι οργανικοί ρύποι του νερού, εκτός από φυσικής προέλευσης, είναι και επικίνδυνες ουσίες από ανθρωπογενείς δραστηριότητες. Στα πλαίσια της προστασίας του υδάτινου περιβάλλοντος και της δημόσιας υγείας εκδόθηκε η πιο πρόσφατη ευρωπαϊκή οδηγία (2013/39/ΕΕ) για τις ουσίες προτεραιότητας με αναθεωρημένα και αυστηρότερα πρότυπα ποιότητας περιβάλλοντος (ΠΠΠ) και με την απόφαση 2015/495/ΕΕ δημιουργήθηκε ο πρώτος κατάλογος επιτήρησης με αναδυόμενους ρύπους του νερού. Οι οδηγίες αυτές προτείνουν νέες στρατηγικές για την αντιμετώπιση της ρύπανσης του νερού και την ανάπτυξη νέων μεθόδων για την συστηματική παρακολούθηση των ρύπων του νερού. Στην παρούσα εργασία αναπτύχθηκε μία πολυδύναμη μέθοδος για τον προσδιορισμό 29 οργανικών ρύπων του νερού (17α-Ethinylestradiol, β-Estradiol, Diclofenac, Alachlor, Atrazine, Chlorfenvinphos, Chlorpyriphos, Diuron, Isoproturon, Simazine, Trifluralin, Pentachlorophenol, 2,4,6-Trichlorophenol, Azinphos-ethyl, Azinphos-methyl, Chlorotoluron, Cyanazine, Diazinon, Dimethoate, Ethion, Fenthion, Linuron, Malathion, Methidathion, Metribuzine, Monuron, Phosalone, Propazine και Triazophos) με LC-MS/MS, εκ των οποίων οι 13 περιλαμβάνονται είτε στις ουσίες προτεραιότητας είτε στον κατάλογο επιτήρησης και οι υπόλοιπες 16 είναι ευρέως χρησιμοποιηθέντα φυτοφάρμακα, πολλά εκ των οποίων έχουν απαγορευθεί. Για τον καθαρισμό του δείγματος και την προσυγκέντρωση των αναλυτών από το νερό, αναπτύχτηκε μέθοδος SPE επιτυγχάνοντας υψηλή ευαισθησία με LODs της τάξης των ng/L.Cyanobacteria, also known as blue-green algae, are present in surface waters and under favorable environmental conditions can form extensive algal blooms and release hazardous toxic compounds (cyanotoxins), posing a significant risk to aquatic ecosystems, drinking water sources and human health. Cyanotoxins comprise a large group of organic compounds, with a variety of physicochemical properties, chemical structures and toxic activity (hepatotoxicity, neurotoxicity, cytotoxicity, dermatotoxicity). Cyanotoxins are classified based on their structure on cyclic peptides (i.e. Microcystins, Nodularins), alkaloids (i.e. Cylindrospermopsin, Anatoxin), lipopolysaccharides and amino acids. Most of the studies on the occurrence of cyanotoxins have been targeted to specific compounds or to variants within a class of cyanotoxins. In this thesis, a fast, simple and sensitive analytical method was developed for the simultaneous determination of multi-class cyanobacterial and algal toxins in water in one run. The target compounds were: Cylindrospermopsin, Anatoxin-a, Nodularin, 12 Microcystins ([D-Asp3]MC-RR, MC-RR, MC-YR, MC-HtyR,[D-Asp3]MC-LR, MC-LR, MC-HilR, MC-WR, MC-LA, MC-LY, MC-LW and MC-LF), Okadaic acid and Domoic acid. Analytes were determined using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in MRM mode. A dual Solid Phase Extraction (SPE) cartridge assembly (two tandem SPE cartridges, Oasis HLB and Hyper PGC) was applied for the efficient extraction of target compounds from water. Moreover, a method was developed for the simultaneous extraction of several cyanotoxins (Cylindrospermopsin, Anatoxin-a and Microcystins) from lyophilized cyanobacterial biomass. During method development several extraction parameters were evaluated. Detection and identification of cyanotoxins Cylindrospermopsin, Anatoxin-a and a wide range of Microcystins in Greek lakes for the first time have demonstrated the applicability and efficiency of the proposed methods. Apart from the natural contaminants of water there are also anthropogenic contaminants with emerging concern. The latest EU Directive on Environmental Quality Standards (EQS Directive 2013/39/EU) has proposed new strategies against water pollution and novel monitoring methods for priority substances. Additionally, the EU Decision 2015/495/EE has established a watch list of emerging contaminants for Union-wide monitoring in the field of water policy. The above directives and decisions implements revised and stricter EQS and making necessary the development and validation of novel multi-residue methods for the determination of micropollutants of water. In this thesis, the development of a novel method for the simultaneous determination of various organic pollutants (i.e. pesticides, endocrine disruptors, pharmaceuticals) in water is presented. The 29 target compounds (17α-Ethinylestradiol, β-Estradiol, Diclofenac, Alachlor, Atrazine, Chlorfenvinphos, Chlorpyriphos, Diuron, Isoproturon, Simazine, Trifluralin, Pentachlorophenol, 2.4.6-Trichlorophenol, Azinphos-ethyl, Azinphos-methyl, Chlorotoluron, Cyanazine, Diazinon, Dimethoate, Ethion, Fenthion, Linuron, Malathion, Methidathion, Metribuzine, Monuron, Phosalone, Propazine and Triazophos), 13 of which are either considered as priority substances or included in watch list and the rest 16 were widely used pesticides many of them have been banned, were determined using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in MRM mode. SPE method, utilizing Oasis HLB cartridges, was developed for sample clean-up and analytes preconcentration from water, enabling high sensitivity with LODs at ng/L level

Myocardial creatine levels do not influence response to acute oxidative stress in isolated perfused heart

Background: Multiple studies suggest creatine mediates anti-oxidant activity in addition to its established role in cellular energy metabolism. The functional significance for the heart has yet to be established, but antioxidant activity could contribute to the cardioprotective effect of creatine in ischaemia/reperfusion injury. Objectives: To determine whether intracellular creatine levels influence responses to acute reactive oxygen species (ROS) exposure in the intact beating heart. We hypothesised that mice with elevated creatine due to over-expression of the creatine transporter (CrT-OE) would be relatively protected, while mice with creatine-deficiency (GAMT KO) would fare worse. Methods and Results: CrT-OE mice were pre-selected for creatine levels 20–100% above wild-type using in vivo 1 H– MRS. Hearts were perfused in isovolumic Langendorff mode and cardiac function monitored throughout. After 20 min equilibration, hearts were perfused with either H2O2 0.5 mM (30 min), or the anti-neoplastic drug doxorubicin 15 mM (100 min). Protein carbonylation, creatine kinase isoenzyme activities and phospho-PKCd expression were quantified in perfused hearts as markers of oxidative damage and apoptotic signalling. Wild-type hearts responded to ROS challenge with a profound decline in contractile function that was ameliorated by co-administration of catalase or dexrazoxane as positive controls. In contrast, the functional deterioration in CrT-OE and GAMT KO hearts was indistinguishable from wildtype controls, as was the extent of oxidative damage and apoptosis. Exogenous creatine supplementation also failed to protect hearts from doxorubicin-induced dysfunction. Conclusions: Intracellular creatine levels do not influence the response to acute ROS challenge in the intact beating heart, arguing against creatine exerting (patho-)physiologically relevant anti-oxidant activity

Dietary Supplementation with Homoarginine Preserves Cardiac Function in a Murine Model of Post-Myocardial Infarction Heart Failure

Low plasma homoarginine (HA) is an emerging biomarker for cardiovascular disease and an independent predictor of mortality in patients with heart failure. Plasma levels appear to reflect cardiac dysfunction, positively correlating with ejection fraction and inversely with circulating brain natriuretic peptide. However, whether this outcome is a bystander or cause-and-effect has yet to be established. Within the context of stroke, a direct causal relationship has been inferred because normal mice pretreated with 14 mg/L HA had a smaller stroke size. In the present study, we show for the first time that dietary supplementation with HA improves cardiac function in the setting of chronic heart failure, suggesting a novel preventive strategy and inferring that low HA levels may be inherently detrimental because of a loss of this effect

Microbes and us: microbiology literacy in Greece

Microbes are ubiquitous and provide numerous services to humans and our planet. However, a query arises as to whether these microbial services are valued by the general public especially after unprecedented conditions like the COVID-19 pandemic. In this context a survey was conducted to investigate the concept of microbe in Greece. Thematic analysis of 672 anonymous responses (age range 4–75yo) received for the open-ended prompt “What is the first thing that comes to mind when you hear the word microbe?” revealed five thematic categories: Negative emotions, Fuzzy associations, Biology, Entities and Health. Almost 80% of responses fell under “Biology” and “Health” and the general pattern of answers was the same across all age groups. Microbes took a variety of forms in the minds of respondents, however, the concept of “microbe” seems to be more unshaped at younger ages (4–11yo), as revealed in children's language choices. Overall, the often-negative perception of microorganisms seems to be confirmed in this study. Although this research was limited to participants from Greece, it remains relevant to other countries around the world as well. We discuss the reasons behind this negative perception and offer suggestions for reversing it

Synergistic effect on cardiac energetics by targeting the creatine kinase system: in vivo application of high-resolution 31P-CMRS in the mouse

Background Phosphorus cardiovascular magnetic resonance spectroscopy (31P-CMRS) has emerged as an important tool for the preclinical assessment of myocardial energetics in vivo. However, the high rate and diminutive size of the mouse heart is a challenge, resulting in low resolution and poor signal-to-noise. Here we describe a refined high-resolution 31P-CMRS technique and apply it to a novel double transgenic mouse (dTg) with elevated myocardial creatine and creatine kinase (CK) activity. We hypothesised a synergistic effect to augment energetic status, evidenced by an increase in the ratio of phosphocreatine-to-adenosine-triphosphate (PCr/ATP). Methods and results Single transgenic Creatine Transporter overexpressing (CrT-OE, n = 7) and dTg mice (CrT-OE and CK, n = 6) mice were anaesthetised with isoflurane to acquire 31P-CMRS measurements of the left ventricle (LV) utilising a two-dimensional (2D), threefold under-sampled density-weighted chemical shift imaging (2D-CSI) sequence, which provided high-resolution data with nominal voxel size of 8.5 µl within 70 min. (1H-) cine-CMR data for cardiac function assessment were obtained in the same imaging session. Under a separate examination, mice received invasive haemodynamic assessment, after which tissue was collected for biochemical analysis. Myocardial creatine levels were elevated in all mouse hearts, but only dTg exhibited significantly elevated CK activity, resulting in a 51% higher PCr/ATP ratio in heart (3.01 ± 0.96 vs. 2.04 ± 0.57—mean ± SD; dTg vs. CrT-OE), that was absent from adjacent skeletal muscle. No significant differences were observed for any parameters of LV structure and function, confirming that augmentation of CK activity does not have unforeseen consequences for the heart. Conclusions We have developed an improved 31P-CMRS methodology for the in vivo assessment of energetics in the murine heart which enabled high-resolution imaging within acceptable scan times. Mice over-expressing both creatine and CK in the heart exhibited a synergistic elevation in PCr/ATP that can now be tested for therapeutic potential in models of chronic heart failure

Chronic creatine kinase deficiency eventually leads to congestive heart failure, but severity is dependent on genetic background, gender and age

The creatine kinase (CK) energy transport and buffering system supports cardiac function at times of high demand and is impaired in the failing heart. Mice deficient in muscle- and mitochondrial-CK (M/Mt-CK(−/−)) have previously been described, but exhibit an unexpectedly mild phenotype of compensated left ventricular (LV) hypertrophy. We hypothesised that heart failure would develop with age and performed echocardiography and LV haemodynamics at 1 year. Since all previous studies have utilised mice with a mixed genetic background, we backcrossed for >10 generations on to C57BL/6, and repeated the in vivo investigations. Male M/Mt-CK(−/−) mice on the mixed genetic background developed congestive heart failure as evidenced by significantly elevated end-diastolic pressure, impaired contractility, LV dilatation, hypertrophy and pulmonary congestion. Female mice were less severely affected, only showing trends for these parameters. After backcrossing, M/Mt-CK(−/−) mice had LV dysfunction consisting of impaired isovolumetric pressure changes and reduced contractile reserve, but did not develop congestive heart failure. Body weight was lower in knockout mice as a consequence of reduced total body fat. LV weight was not significantly elevated in relation to other internal organs and gene expression of LVH markers was normal, suggesting an absence of hypertrophy. In conclusion, the consequences of CK deficiency are highly dependent on genetic modifiers, gender and age. However, the observation that a primary defect in CK can, under the right conditions, result in heart failure suggests that impaired CK activity in the failing heart could contribute to disease progression. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00395-012-0276-2) contains supplementary material, which is available to authorized users

Homoarginine and creatine deficiency do not exacerbate murine ischaemic heart failure

Aims: Low levels of homoarginine and creatine are associated with heart failure severity in humans, but it is unclear to what extent they contribute to pathophysiology. Both are synthesized via L-arginine:glycine amidinotransferase (AGAT), such that AGAT−/− mice have a combined creatine and homoarginine deficiency. We hypothesized that this would be detrimental in the setting of chronic heart failure. Methods and results: Study 1: homoarginine deficiency—female AGAT−/− and wild-type mice were given creatine-supplemented diet so that both had normal myocardial creatine levels, but only AGAT−/− had low plasma homoarginine. Myocardial infarction (MI) was surgically induced and left ventricular (LV) structure and function assessed at 6–7 weeks by in vivo imaging and haemodynamics. Study 2: homoarginine and creatine-deficiency—as before, but AGAT−/− mice were given creatine-supplemented diet until 1 week post-MI, when 50% were changed to a creatine-free diet. Both groups therefore had low homoarginine levels, but one group also developed lower myocardial creatine levels. In both studies, all groups had LV remodelling and dysfunction commensurate with the development of chronic heart failure, for example, LV dilatation and mean ejection fraction <20%. However, neither homoarginine deficiency alone or in combination with creatine deficiency had a significant effect on mortality, LV remodelling, or on any indices of contractile and lusitropic function. Conclusions: Low levels of homoarginine and creatine do not worsen chronic heart failure arguing against a major causative role in disease progression. This suggests that it is unnecessary to correct hArg deficiency in patients with heart failure, although supra-physiological levels may still be beneficial

Cardiac function and energetics in mice with combined genetic augmentation of creatine and creatine kinase activity

Improving energy provision in the failing heart by augmenting the creatine kinase (CK) system is a desirable therapeutic target. However, over-expression of the creatine transporter (CrT-OE) has shown that very high creatine levels result in cardiac hypertrophy and dysfunction. We hypothesise this is due to insufficient endogenous CK activity to maintain thermodynamically favourable metabolite ratios. If correct, then double transgenic mice (dTg) overexpressing both CrT and the muscle isoform of CK (CKM-OE) would rescue the adverse phenotype. In Study 1, overexpressing lines were crossed and cardiac function assessed by invasive haemodynamics and echocardiography. This demonstrated that CKM-OE was safe, but too few hearts had creatine in the toxic range. In Study 2, a novel CrT-OE line was generated with higher, homogeneous, creatine levels and phenotyped as before. Myocardial creatine was 4-fold higher in CrT-OE and dTg hearts compared to wildtype and was associated with hypertrophy and contractile dysfunction. The inability of dTg hearts to rescue this phenotype was attributed to downregulation of CK activity, as occurs in the failing heart. Nevertheless, combining both studies in a linear regression analysis suggests a modest positive effect of CKM over a range of creatine concentrations. In conclusion, we confirm that moderate elevation of creatine is well tolerated, but very high levels are detrimental. Correlation analysis lends support to the theory that this may be a consequence of limited CK activity. Future studies should focus on preventing CKM downregulation to unlock the potential synergy of augmenting both creatine and CK in the heart