Novel AlkB Dioxygenases—Alternative Models for In Silico and In Vivo Studies

Background: ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a direct, single-protein repair system, protecting cellular DNA and RNA against the cytotoxic and mutagenic activity of alkylating agents, chemicals significantly contributing to tumor formation and used in cancer therapy. In silico analysis and in vivo studies have shown the existence of AlkB homologs in almost all organisms. Nine AlkB homologs (ALKBH1–8 and FTO) have been identified in humans. High ALKBH levels have been found to encourage tumor development, questioning the use of alkylating agents in chemotherapy. The aim of this work was to assign biological significance to multiple AlkB homologs by characterizing their activity in the repair of nucleic acids in prokaryotes and their subcellular localization in eukaryotes. Methodology and Findings: Bioinformatic analysis of protein sequence databases identified 1943 AlkB sequences with eight new AlkB subfamilies. Since Cyanobacteria and Arabidopsis thaliana contain multiple AlkB homologs, they were selected as model organisms for in vivo research. Using E. coli alkB2 mutant and plasmids expressing cyanobacterial AlkBs, we studied the repair of methyl methanesulfonate (MMS) and chloroacetaldehyde (CAA) induced lesions in ssDNA, ssRNA, and genomic DNA. On the basis of GFP fusions, we investigated the subcellular localization of ALKBHs in A. thaliana and established its mostly nucleo-cytoplasmic distribution. Some of the ALKBH proteins were found to change their localization upon MMS treatment. Conclusions: Our in vivo studies showed highly specific activity of cyanobacterial AlkB proteins towards lesions and nucleic acid type. Subcellular localization and translocation of ALKBHs in A. thaliana indicates a possible role for these proteins in the repair of alkyl lesions. We hypothesize that the multiplicity of ALKBHs is due to their involvement in the metabolism of nucleo-protein complexes; we find their repair by ALKBH proteins to be economical and effective alternative to degradation and de novo synthesis

Lethal and mutagenic properties of MMS-generated DNA lesions in Escherichia coli cells deficient in BER and AlkB-directed DNA repair.

Methylmethane sulphonate (MMS), an S(N)2-type alkylating agent, generates DNA methylated bases exhibiting cytotoxic and mutagenic properties. Such damaged bases can be removed by a system of base excision repair (BER) and by oxidative DNA demethylation catalysed by AlkB protein. Here, we have shown that the lack of the BER system and functional AlkB dioxygenase results in (i) increased sensitivity to MMS, (ii) elevated level of spontaneous and MMS-induced mutations (measured by argE3 --> Arg(+) reversion) and (iii) induction of the SOS response shown by visualization of filamentous growth of bacteria. In the xth nth nfo strain additionally mutated in alkB gene, all these effects were extreme and led to 'error catastrophe', resulting from the presence of unrepaired apurinic/apyrimidinic (AP) sites and 1-methyladenine (1meA)/3-methylcytosine (3meC) lesions caused by deficiency in, respectively, BER and AlkB dioxygenase. The decreased level of MMS-induced Arg(+) revertants in the strains deficient in polymerase V (PolV) (bearing the deletion of the umuDC operon), and the increased frequency of these revertants in bacteria overproducing PolV (harbouring the pRW134 plasmid) indicate the involvement of PolV in the error-prone repair of 1meA/3meC and AP sites. Comparison of the sensitivity to MMS and the induction of Arg(+) revertants in the double nfo alkB and xth alkB, and the quadruple xth nth nfo alkB mutants showed that the more AP sites there are in DNA, the stronger the effect of the lack of AlkB protein. Since the sum of MMS-induced Arg(+) revertants in xth, nfo and nth xth nfo and alkB mutants is smaller than the frequency of these revertants in the BER(-) alkB(-) strain, we consider two possibilities: (i) the presence of AP sites in DNA results in relaxation of its structure that facilitates methylation and (ii) additional AP sites are formed in the BER(-) alkB(-) mutants

Thrombus aspiration in ST elevation myocardial infarction: comparative efficacy in patients treated early and late after onset of symptoms

Background Restoration of myocardial perfusion is the goal of percutaneous coronary intervention (PCI) in patients with ST elevation myocardial infarction. A major predictor of no-reflow is the increasing time to treatment (TTT). Thrombus aspiration (TA) is reported to improve myocardial reperfusion as compared with standard PCI (SP). Objective To investigate the influence of TTT on TA efficacy. Design Pooled analysis of individual patients' data of three prospective randomised trials comparing TA and SP. Patients A total of 299 patients (150 in TA group and 149 in SP group) entered the study. The study population was divided into three subgroups according to the TTT: <= 3 h (short TTT subgroup), >3 h to <= 6 h (intermediate TTT subgroup), >6 h to <= 12 h (long TTT subgroup). Main outcome measures The goal of the study was the comparison of optimal myocardial reperfusion, defined as the combination of myocardial blush grade 2 or 3 at post-PCI angiography and ST resolution more than 70% at post-PCI ECG, between SP and TA according to TTT. Results In the SP group, increasing TTT was associated with a decreased rate of optimal reperfusion (27.4% vs 17.9% vs 10%, p for trend=0.06), whereas in the TA group the same trend was not seen (40.9% vs 33.8% vs 50%, p for trend=0.93). In a multivariate logistic regression model, a significant interaction (p=0.04) between time to treatment and thrombus aspiration was observed. Conclusions TA limits the adverse effects of TTT prolongation on myocardial reperfusion

Thrombus aspiration in ST elevation myocardial infarction: comparative efficacy in patients treated early and late after onset of symptoms

Background Restoration of myocardial perfusion is the goal of percutaneous coronary intervention (PCI) in patients with ST elevation myocardial infarction. A major predictor of no-reflow is the increasing time to treatment (TTT). Thrombus aspiration (TA) is reported to improve myocardial reperfusion as compared with standard PCI (SP). Objective To investigate the influence of TTT on TA efficacy. Design Pooled analysis of individual patients' data of three prospective randomised trials comparing TA and SP. Patients A total of 299 patients (150 in TA group and 149 in SP group) entered the study. The study population was divided into three subgroups according to the TTT: 3 h to 6 h to <= 12 h (long TTT subgroup). Main outcome measures The goal of the study was the comparison of optimal myocardial reperfusion, defined as the combination of myocardial blush grade 2 or 3 at post-PCI angiography and ST resolution more than 70% at post-PCI ECG, between SP and TA according to TTT. Results In the SP group, increasing TTT was associated with a decreased rate of optimal reperfusion (27.4% vs 17.9% vs 10%, p for trend=0.06), whereas in the TA group the same trend was not seen (40.9% vs 33.8% vs 50%, p for trend=0.93). In a multivariate logistic regression model, a significant interaction (p=0.04) between time to treatment and thrombus aspiration was observed. Conclusions TA limits the adverse effects of TTT prolongation on myocardial reperfusion

Thrombus aspiration in ST elevation myocardial infarction: comparative efficacy in patients treated early and late after onset of symptoms

Background Restoration of myocardial perfusion is the goal of percutaneous coronary intervention (PCI) in patients with ST elevation myocardial infarction. A major predictor of no-reflow is the increasing time to treatment (TTT). Thrombus aspiration (TA) is reported to improve myocardial reperfusion as compared with standard PCI (SP). Objective To investigate the influence of TTT on TA efficacy. Design Pooled analysis of individual patients' data of three prospective randomised trials comparing TA and SP. Patients A total of 299 patients (150 in TA group and 149 in SP group) entered the study. The study population was divided into three subgroups according to the TTT: &lt;= 3 h (short TTT subgroup), &gt;3 h to &lt;= 6 h (intermediate TTT subgroup), &gt;6 h to &lt;= 12 h (long TTT subgroup). Main outcome measures The goal of the study was the comparison of optimal myocardial reperfusion, defined as the combination of myocardial blush grade 2 or 3 at post-PCI angiography and ST resolution more than 70% at post-PCI ECG, between SP and TA according to TTT. Results In the SP group, increasing TTT was associated with a decreased rate of optimal reperfusion (27.4% vs 17.9% vs 10%, p for trend=0.06), whereas in the TA group the same trend was not seen (40.9% vs 33.8% vs 50%, p for trend=0.93). In a multivariate logistic regression model, a significant interaction (p=0.04) between time to treatment and thrombus aspiration was observed. Conclusions TA limits the adverse effects of TTT prolongation on myocardial reperfusion

CARESS-in-AMI (Combined Abciximab RE-teplase Stent Study in Acute Myocardial Infarction) Investigators. Immediate angioplasty versus standard therapy with rescue angioplasty after thrombolysis in the Combined Abciximab REteplase Stent Study in Acute Myocardial Infarction (CARESS-in-AMI): an open, prospective, randomised, multicentre trial.

BACKGROUND: Thrombolysis remains the treatment of choice in ST-segment elevation myocardial infarction (STEMI) when primary percutaneous coronary intervention (PCI) cannot be done within 90 min. However, the best subsequent management of patients after thrombolytic therapy remains unclear. To assess the best management, we randomised patients with STEMI treated by thrombolysis and abciximab at a non-interventional hospital to immediate transfer for PCI, or to standard medical therapy with transfer for rescue angioplasty. METHODS: 600 patients aged 75 years or younger with one or more high-risk features (extensive ST-segment elevation, new-onset left bundle branch block, previous myocardial infarction, Killip class >2, or left ventricular ejection fraction < or =35%) in hospitals in France, Italy, and Poland were treated with half-dose reteplase, abciximab, heparin, and aspirin, and randomly assigned to immediate transfer to the nearest interventional centre for PCI, or to management in the local hospital with transfer only in case of persistent ST-segment elevation or clinical deterioration. The primary outcome was a composite of death, reinfarction, or refractory ischaemia at 30 days, and analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number 00220571. FINDINGS: Of the 299 patients assigned to immediate PCI, 289 (97.0%) underwent angiography, and 255 (85.6%) received PCI. Rescue PCI was done in 91 patients (30.3%) in the standard care/rescue PCI group. The primary outcome occurred in 13 patients (4.4%) in the immediate PCI group compared with 32 (10.7%) in the standard care/rescue PCI group (hazard ratio 0.40; 95% CI 0.21-0.76, log rank p=0.004). Major bleeding was seen in ten patients in the immediate group and seven in the standard care/rescue group (3.4%vs 2.3%, p=0.47). Strokes occurred in two patients in the immediate group and four in the standard care/rescue group (0.7%vs 1.3%, p=0.50). INTERPRETATION: Immediate transfer for PCI improves outcome in high-risk patients with STEMI treated at a non-interventional centre with half-dose reteplase and abciximab