Enhancement of the cytotoxic potential of the mixed EGFR and DNA-targeting 'combi-molecule' ZRBA1 against human solid tumour cells by a bis-quinazoline-based drug design approach

ZRBA1 is a quinazoline-based molecule termed 'combi-molecule' designed to block the epidermal growth factor receptor (EGFR) and further degrade to FD105, an EGFR inhibitor plus a DNA-alkylating agent. To augment the potency of ZRBA1, we designed JDE52, a bistriazene that, following degradation, was 'programmed' to yield higher concentrations of the free inhibitor FD105 and a more cytotoxic bifunctional DNA-damaging species. The results indicated that JDE52 was capable of inducing significant blockade of EGFR phosphorylation, DNA strand breaks and interstrand cross-links in human cells. The fluorescent property of FD105, the secondary inhibitor that both JDE52 and ZRBA1 are capable of releasing, has permitted the analysis of its levels in tumour cells by ultraviolet flow cytometry. It was found that JDE52 was indeed capable of significantly releasing higher levels of fluorescence (P<0.05) in human tumour cells when compared with ZRBA1. Apoptosis was triggered by JDE52 at a faster rate than ZRBA1 and led to higher levels of cell killing. The results in toto suggest that the superior potency of JDE52, when compared with ZRBA1, may be imputed to mechanisms associated with the generation of higher intracellular concentrations of FD105 and to the induction of DNA cross-links. These combined mechanisms (blockade of EGFR-tyrosine kinase and induction of cross-links) contributed to an accelerated rate of apoptosis by JDE52. This study conclusively demonstrated that designing molecules as prodrugs of high levels of quinazoline inhibitors of EGFR and bifunctional DNA cross-linking species is a valid strategy to enhance the potency of mixed EGFR-DNA-targeting combi-molecules.     

Targeted gene expression profiling in Leishmania braziliensis and Leishmania guyanensis parasites isolated from Brazilian patients with different antimonial treatment outcomes

In Brazil, cutaneous leishmaniasis represents a serious public health problem, and chemotherapy is an important element of the clinical management of this disease. However, treatment efficacy is variable, a phenomenon that might be due to host and parasite (e.g., drug resistance) factors. To better understand the possible contribution of parasite factors to this phenomenon, we characterised 12 Leishmania braziliensis (LB) and 25 Leishmania guyanensis (LG) isolates collected from patients experiencing different antimonial treatment outcomes. For each isolate, promastigote cultures were grown in duplicate and were harvested at the late-log and stationary phases of growth. The RNA expression profiles of six genes encoding proteins with roles in antimony metabolism (AQP1, MRPA, GSH1, GSH2, TRYR and TDR1) were assessed by means of real-time quantitative PCR. Molecular data were compared to the clinical phenotypes. Within LB, we did not find statistically significant differences in the expression levels of the examined genes among isolates from patients with different treatment outcomes. In LG, GSH1 (encoding gamma-glutamylcysteine synthetase, γ-GCS) was overexpressed in therapeutic failure isolates regardless of the growth curve phase. This finding reveals the predictive potential of promastigote expression curves for the prognosis of cutaneous leishmaniasis caused by LG in Brazil.     

What is the level of evidence for combined cardiac resynchronization and defibrillation therapy in heart failure? reply

In the letter, Doctors Lam and Owen offer some very relevantcomments on the recommendations for cardiac resynchronizationcombined with defibrillation therapy (CRT-D)     

Variation in dopamine genes influences responsivity of the human reward system

In humans, dopamine neurotransmission is influenced by functional polymorphisms in the dopamine transporter (DAT1) and catechol-O-methyltransferase (COMT) genes. Here, we used event-related functional magnetic resonance imaging to directly investigate the neurofunctional effects of the Val¹⁵⁸Met COMT and variable number of tandem repeat DAT1 polymorphisms on distinct components of the reward system in humans. The results revealed a main effect of COMT genotype in the ventral striatum and lateral prefrontal cortex during reward anticipation (P < 0.001, uncorrected) and in the orbitofrontal cortex at the time of reward delivery (P < 0.005), met/met individuals exhibiting the highest activation. The main effect of DAT1 genotype was seen in robust blood-oxygen-level-dependent response differences in the caudate nucleus and ventral striatum during reward anticipation (P < 0.001) and in the lateral prefrontal cortex and midbrain at the time of reward delivery, with carriers of the DAT1 9-repeat allele showing the highest activity. Moreover, an interaction between the COMT and DAT1 genes was found in the ventral striatum and lateral prefrontal cortex during reward anticipation and in the lateral prefrontal and orbitofrontal cortices as well as in the midbrain at the time of reward delivery, with carriers of the DAT1 9-repeat allele and COMT met/met allele exhibiting the highest activation, presumably reflecting functional change consequent to higher synaptic dopamine availability. Taken together, these results indicate that genetically influenced variations in dopamine transmission modulate the response of brain regions involved in anticipation and reception of rewards and suggest that these responses may contribute to individual differences in reward-seeking behavior and in predisposition to neuropsychiatric disorders.     

Prophylactic plasma exchange in CD46-associated atypical haemolytic uremic syndrome

Patients with atypical haemolytic uremic syndrome (aHUS) with a mutation in the gene encoding membrane cofactor protein (CD46) are known to have a better prognosis than those with mutations in factor H (CFH) or factor I (CFI), but a small number of the former still proceed to end-stage renal failure. Plasma therapy (PE) is the recommended approach to treat both acute episodes and prevent recurrences in aHUS, but studies have yet to show PE efficacy in aHUS associated with a CD46 mutation. The factors determining failure to treatment are not clear and may be related to the mutation involved or to insufficient treatment. Our experience of PE in a family of three sisters with CFH-associated aHUS suggests that intensive and prophylactic PE allows renal function to be maintained in both native kidneys and allografts. The success of this strategy has led us to use it in all cases of aHUS. Here, we describe the effect of this strategy in a child with aHUS and a CD46 mutation. The initial episode was treated with daily PE, resulting in the recovery of renal function. However, over the next 4 years, there was a progressive decline in renal function to end-stage renal failure, with evidence of an on-going thrombotic microangiopathy despite continuous prophylactic PE. Prophylactic PE does not influence the natural course of aHUS and CD46 mutation.     

Primary Health Care for Hypertension by Nurses in Rural and Urban Sub-Saharan Africa

To implement a nurse-led protocol for the care of hypertension, 5 clinics were established in Yaounde (urban) and Bafut (rural) in Cameroon. International guidelines were adapted and 10 nurses were trained. The initial cohort of patients was referred from a field survey. The program proceeded for 26 months and 454 patients (45% urban) were registered in the clinics. Relative to urban participants, rural participants were more often women (59% vs 45%, P=.002) and less likely to have diabetes (7.2% vs 41.2%, P<.001). Between baseline and final visits, systolic and diastolic blood pressures dropped by 11.7 mm Hg (95% confidence interval, 8.9–14.4) and 7.8 (95% confidence interval, 5.9–9.6), respectively (P<.001). These changes were consistent in subgroups and after adjustment. Most dropouts occurred around the initial visit and among urban participants and nondiabetics. Nurse-led clinics are effective for improving hypertension care in these settings and require implementation and validation through controlled trials.