Investigation of tumor hypoxia using a two-enzyme system for in vitro generation of oxygen deficiency

<p>Abstract</p> <p>Background</p> <p>Oxygen deficiency in tumor tissue is associated with a malign phenotype, characterized by high invasiveness, increased metastatic potential and poor prognosis. Hypoxia chambers are the established standard model for <it>in vitro </it>studies on tumor hypoxia. An enzymatic hypoxia system (GOX/CAT) based on the use of glucose oxidase (GOX) and catalase (CAT) that allows induction of stable hypoxia for <it>in vitro </it>approaches more rapidly and with less operating expense has been introduced recently. Aim of this work is to compare the enzymatic system with the established technique of hypoxia chamber in respect of gene expression, glucose metabolism and radioresistance, prior to its application for <it>in vitro </it>investigation of oxygen deficiency.</p> <p>Methods</p> <p>Human head and neck squamous cell carcinoma HNO97 cells were incubated under normoxic and hypoxic conditions using both hypoxia chamber and the enzymatic model. Gene expression was investigated using Agilent microarray chips and real time PCR analysis. ¹⁴C-fluoro-deoxy-glucose uptake experiments were performed in order to evaluate cellular metabolism. Cell proliferation after photon irradiation was investigated for evaluation of radioresistance under normoxia and hypoxia using both a hypoxia chamber and the enzymatic system.</p> <p>Results</p> <p>The microarray analysis revealed a similar trend in the expression of known HIF-1 target genes between the two hypoxia systems for HNO97 cells. Quantitative RT-PCR demonstrated different kinetic patterns in the expression of carbonic anhydrase IX and lysyl oxidase, which might be due to the faster induction of hypoxia by the enzymatic system. ¹⁴C-fluoro-deoxy-glucose uptake assays showed a higher glucose metabolism under hypoxic conditions, especially for the enzymatic system. Proliferation experiments after photon irradiation revealed increased survival rates for the enzymatic model compared to hypoxia chamber and normoxia, indicating enhanced resistance to irradiation. While the GOX/CAT system allows independent investigation of hypoxia and oxidative stress, care must be taken to prevent acidification during longer incubation.</p> <p>Conclusion</p> <p>The results of our study indicate that the enzymatic model can find application for <it>in vitro </it>investigation of tumor hypoxia, despite limitations that need to be considered in the experimental design.</p

Genetic Reconstruction of Protozoan rRNA Decoding Sites Provides a Rationale for Paromomycin Activity against Leishmania and Trypanosoma

Aminoglycoside antibiotics target the ribosomal decoding A-site and are active against a broad spectrum of bacteria. These compounds bind to a highly conserved stem-loop-stem structure in helix 44 of bacterial 16S rRNA. One particular aminoglycoside, paromomycin, also shows potent antiprotozoal activity and is used for the treatment of parasitic infections, e.g. by Leishmania spp. The precise drug target is, however, unclear; in particular whether aminoglycoside antibiotics target the cytosolic and/or the mitochondrial protozoan ribosome. To establish an experimental model for the study of protozoan decoding-site function, we constructed bacterial chimeric ribosomes where the central part of bacterial 16S rRNA helix 44 has been replaced by the corresponding Leishmania and Trypanosoma rRNA sequences. Relating the results from in-vitro ribosomal assays to that of in-vivo aminoglycoside activity against Trypanosoma brucei, as assessed in cell cultures and in a mouse model of infection, we conclude that aminoglycosides affect cytosolic translation while the mitochondrial ribosome of trypanosomes is not a target for aminoglycoside antibiotics

Vasodilator myocardial perfusion cardiac magnetic resonance imaging is superior to dobutamine stress echocardiography in the detection of relevant coronary artery Stenosis: a systematic review and meta-analysis on their diagnostic accuracy

Objectives: Guideline recommendations for patients with either a high or a low risk of obstructive coronary artery disease (CAD) are clear. However, the evidence for initial risk stratification in patients with an intermediate risk of CAD is still unclear, despite the availability of multiple non-invasive assessment strategies. The aim of this study was to synthesize the evidence for this population to provide more informed recommendations. Background: A meta-analysis was performed to systematically assess the diagnostic accuracy of vasodilator myocardial perfusion cardiovascular magnetic resonance imaging (pCMR) and dobutamine stress echocardiography (DSE) for the detection of relevant CAD. In contrast to previous work, this meta-analysis follows rigorous selection criteria in regards to the risk stratification and a narrowly prespecified definition of their invasive reference tests, resulting in unprecedentedly informative results for this reference group. Data Collection and Analysis: From the 5,634 studies identified, 1,306 relevant articles were selected after title screening and further abstract screening left 865 studies for full-text review. Of these, 47 studies fulfilled all inclusion criteria resulting in a total sample size of 4,742 patients. Results: pCMR studies showed a superior sensitivity [0.88 (95% confidence interval (CI): 0.85–0.90) vs. 0.72 (95% CI: 0.61–0.81)], diagnostic odds ratio (DOR) [38 (95% CI: 29–49) vs. 20 (95% CI: 9–46)] and an augmented post-test probability [negative likelihood ratio (LR) of 0.14 (95% CI: 0.12–0.18) vs. 0.31 (95% CI: 0.21, 0.46)] as compared to DSE. Specificity was statistically indifferent [0.84 (95% CI: 0.81–0.87) vs. 0.89 (95% CI: 0.83–0.93)]. Conclusion: The results of this systematic review and meta-analysis suggest that pCMR has a superior diagnostic test accuracy for relevant CAD compared to DSE. In patients with intermediate risk of CAD only pCMR can reliably rule out relevant stenosis. In this risk cohort, pCMR can be offered for initial risk stratification and guidance of further invasive treatment as it also rules in relevant CAD

Anaemia is associated with severe RBC dysfunction and a reduced circulating NO pool: vascular and cardiac eNOS are crucial for the adaptation to anaemia

Anaemia is frequently present in patients with acute myocardial infarction (AMI) and contributes to an adverse prognosis. We hypothesised that, besides reduced oxygen carrying capacity, anaemia is associated with (1) red blood cell (RBC) dysfunction and a reduced circulating nitric oxide (NO) pool, (2) compensatory enhancement of vascular and cardiac endothelial nitric oxide synthase (eNOS) activity, and (3) contribution of both, RBC dysfunction and reduced circulatory NO pool to left ventricular (LV) dysfunction and fatal outcome in AMI. In mouse models of subacute and chronic anaemia from repeated mild blood loss the circulating NO pool, RBC, cardiac and vascular function were analysed at baseline and in reperfused AMI. In anaemia, RBC function resulted in profound changes in membrane properties, enhanced turnover, haemolysis, dysregulation of intra-erythrocytotic redox state, and RBC-eNOS. RBC from anaemic mice and from anaemic patients with acute coronary syndrome impaired the recovery of contractile function of isolated mouse hearts following ischaemia/reperfusion. In anaemia, the circulating NO pool was reduced. The cardiac and vascular adaptation to anaemia was characterised by increased arterial eNOS expression and activity and an eNOS-dependent increase of end-diastolic left ventricular volume. Endothelial dysfunction induced through genetic or pharmacologic reduction of eNOS-activity abrogated the anaemia-induced cardio-circulatory compensation. Superimposed AMI was associated with decreased survival. In summary, moderate blood loss anaemia is associated with severe RBC dysfunction and reduced circulating NO pool. Vascular and cardiac eNOS are crucial for the cardio-circulatory adaptation to anaemia. RBC dysfunction together with eNOS dysfunction may contribute to adverse outcomes in AMI