Phosphoenolpyruvate carboxylase dentified as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism

Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in thePlasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10Δpepc), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10Δpepc had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using 13C-U-D-glucose and 13C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10Δpepc and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of 13C,15N-U-glutamine was similar in both parasite lines, although the flux was lower in D10Δpepc; it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery

Central nervous system relapse in patients with breast cancer is associated with advanced stages, with the presence of circulating occult tumor cells and with the HER2/neu status

INTRODUCTION: To evaluate the incidence of central nervous system (CNS) involvement in patients with breast cancer treated with a taxane-based chemotherapy regimen and to determine predictive factors for CNS relapse. METHODS: The medical files of patients with early breast cancer (n = 253) or advanced stage breast cancer (n = 239) as well of those with other solid tumors (n = 336) treated with or without a taxane-based chemotherapy regimen during a 42-month period were reviewed. HER2/neu overexpression was identified by immunohistochemistry, whereas cytokeratin 19 (CK-19) mRNA-positive circulating tumor cells (CTCs) in the peripheral blood were identified by real-time PCR. RESULTS: The incidence of CNS relapse was similar in patients suffering from breast cancer or other solid tumors (10.4% and 11.4%, respectively; P = 0.517). The incidence of CNS relapse was significantly higher in breast cancer patients with advanced disease (P = 0.041), visceral disease and bone disease (P = 0.036), in those who were treated with a taxane-containing regimen (P = 0.024), in those with HER2/neu-overexpressing tumors (P = 0.022) and, finally, in those with detectable CK-19 mRNA-positive CTCs (P = 0.008). Multivariate analysis revealed that the stage of disease (odds ratio, 0.23; 95% confidence interval, 0.007–0.23; P = 0.0001), the HER2/neu status (odds ratio, 29.4; 95% confidence interval, 7.51–101.21; P = 0.0001) and the presence of CK-19 mRNA-positive CTCs (odds ratio, 8.31; 95% confidence interval, 3.97–12.84; P = 0.001) were independent predictive factors for CNS relapse. CONCLUSION: CNS relapses are common among breast cancer patients treated with a taxane-based chemotherapy regimen, patients with HER2/neu-positive tumor and patients with CK-19 mRNA-positive CTCs

Transcription and Expression of Plasmodium falciparum Histidine-Rich Proteins in Different Stages and Strains: Implications for Rapid Diagnostic Tests

Background: Although rapid diagnostic tests (RDTs) for Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2) are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates. Methods: Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle. Results: Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed. Conclusions: The levels of transcription of pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs

Aerosolized Delivery of Antifungal Agents

Pulmonary infections caused by Aspergillus species are associated with significant morbidity and mortality in immunocompromised patients. Although the treatment of pulmonary fungal infections requires the use of systemic agents, aerosolized delivery is an attractive option in prevention because the drug can concentrate locally at the site of infection with minimal systemic exposure. Current clinical evidence for the use of aerosolized delivery in preventing fungal infections is limited to amphotericin B products, although itraconazole, voriconazole, and caspofungin are under investigation. Based on conflicting results from clinical trials that evaluated various amphotericin B formulations, the routine use of aerosolized delivery cannot be recommended. Further research with well-designed clinical trials is necessary to elucidate the therapeutic role and risks associated with aerosolized delivery of antifungal agents. This article provides an overview of aerosolized delivery systems, the intrapulmonary pharmacokinetic properties of aerosolized antifungal agents, and key findings from clinical studies