Unraveling the mode of action of the antimalarial choline analog G25 in Plasmodium falciparum and Saccharomyces cerevisiae

Pharmacological studies have indicated that the choline analog G25 is a potent inhibitor of Plasmodium falciparum growth in vitro and in vivo. Although choline transport has been suggested to be the target of G25, the exact mode of action of this compound is not known. Here we show that, similar to its effects on P. falciparum, G25 prevents choline entry into Saccharomyces cerevisiae cells and inhibits S. cerevisiae growth. However, we show that the uptake of this compound is not mediated by the choline carrier Hnm1. An hnm1Delta yeast mutant, which lacks the only choline transporter gene HNM1, was not altered in the transport of a labeled analog of this compound. Eleven yeast mutants lacking genes involved in different steps of phospholipid biosynthesis were analyzed for their sensitivity to G25. Four mutants affected in the de novo cytidyldiphosphate-choline-dependent phosphatidylcholine biosynthetic pathway and, surprisingly, a mutant strain lacking the phosphatidylserine decarboxylase-encoding gene PSD1 (but not PSD2) were found to be highly resistant to this compound. Based on these data for S. cerevisiae, labeling studies in P. falciparum were performed to examine the effect of G25 on the biosynthetic pathways of the major phospholipids phosphatidylcholine and phosphatidylethanolamine. Labeling studies in P. falciparum and in vitro studies with recombinant P. falciparum phosphatidylserine decarboxylase further supported the inhibition of both the de novo phosphatidylcholine metabolic pathway and the synthesis of phosphatidylethanolamine from phosphatidylserine. Together, our data indicate that G25 specifically targets the pathways for synthesis of the two major phospholipids, phosphatidylcholine and phosphatidylethanolamine, to exert its antimalarial activity.     

Lasso regularization for left-censored Gaussian outcome and high-dimensional predictors

Background

Biological assays for the quantification of markers may suffer from a lack of sensitivity and thus from an analytical detection limit. This is the case of human immunodeficiency virus (HIV) viral load. Below this threshold the exact value is unknown and values are consequently left-censored. Statistical methods have been proposed to deal with left-censoring but few are adapted in the context of high-dimensional data.

Methods

We propose to reverse the Buckley-James least squares algorithm to handle left-censored data enhanced with a Lasso regularization to accommodate high-dimensional predictors. We present a Lasso-regularized Buckley-James least squares method with both non-parametric imputation using Kaplan-Meier and parametric imputation based on the Gaussian distribution, which is typically assumed for HIV viral load data after logarithmic transformation. Cross-validation for parameter-tuning is based on an appropriate loss function that takes into account the different contributions of censored and uncensored observations. We specify how these techniques can be easily implemented using available R packages. The Lasso-regularized Buckley-James least square method was compared to simple imputation strategies to predict the response to antiretroviral therapy measured by HIV viral load according to the HIV genotypic mutations. We used a dataset composed of several clinical trials and cohorts from the Forum for Collaborative HIV Research (HIV Med. 2008;7:27-40). The proposed methods were also assessed on simulated data mimicking the observed data.

Results

Approaches accounting for left-censoring outperformed simple imputation methods in a high-dimensional setting. The Gaussian Buckley-James method with cross-validation based on the appropriate loss function showed the lowest prediction error on simulated data and, using real data, the most valid results according to the current literature on HIV mutations.

Conclusions

The proposed approach deals with high-dimensional predictors and left-censored outcomes and has shown its interest for predicting HIV viral load according to HIV mutations.

     

Treatment with Sunitinib Enabled Complete Resection of Massive Lymphadenopathy not Previously Amenable to Excision in a Patient with Renal Cell Carcinoma

We present a case of previously unresectable lymphadenopathy in a patient with renal cell carcinoma treated with sunitinib. Complete resection of a 15-cm left renal cell carcinoma was initially impossible due to massive retroperitoneal disease and encasement of the great vessels and mesenteric vessels. Residual retroperitoneal disease from a radical nephrectomy was treated with the oral, multitargeted receptor tyrosine kinase inhibitor, sunitinib. Tumour shrinkage following five cycles of treatment allowed uncomplicated complete resection of the lymphadenopathy. Follow-up after 6 mo showed no evidence of disease recurrence.     

Prognosis of HIV-1-infected patients up to 5 years after initiation of HAART: collaborative analysis of prospective studies

OBJECTIVE: To estimate the prognosis over 5 years of HIV-1-infected, treatment-naive patients starting HAART, taking into account the immunological and virological response to therapy. DESIGN: A collaborative analysis of data from 12 cohorts in Europe and North America on 20,379 adults who started HAART between 1995 and 2003. METHODS: Parametric survival models were used to predict the cumulative incidence at 5 years of a new AIDS-defining event or death, and death alone, first from the start of HAART and second from 6 months after the start of HAART. Data were analysed by intention-to-continue-treatment, ignoring treatment changes and interruptions. RESULTS: During 61 798 person-years of follow-up, 1005 patients died and an additional 1303 developed AIDS. A total of 10 046 (49%) patients started HAART either with a CD4 cell count of less than 200 cells/microl or with a diagnosis of AIDS. The 5-year risk of AIDS or death (death alone) from the start of HAART ranged from 5.6 to 77% (1.8-65%), depending on age, CD4 cell count, HIV-1-RNA level, clinical stage, and history of injection drug use. From 6 months the corresponding figures were 4.1-99% for AIDS or death and 1.3-96% for death alone. CONCLUSION: On the basis of data collected routinely in HIV care, prognostic models with high discriminatory power over 5 years were developed for patients starting HAART in industrialized countries. A risk calculator that produces estimates for progression rates at years 1 to 5 after starting HAART is available from www.art-cohort-collaboration.org.     

beta3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current

beta3-adrenergic receptor (beta3-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of beta3-AR in the human atrium. Unexpectedly, beta3-AR activation increased human atrial tissue contractility and stimulated the L-type Ca2+ channel current (I Ca,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The I(Ca,L) and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective beta3-AR agonists, SR58611 and BRL37344, and a beta3-AR partial agonist, CGP12177, stimulated I(Ca,L) in HAMs with nanomolar potency and a 60%-90% efficacy compared with isoprenaline. The beta3-AR agonists also increased contractility but with a much lower efficacy (approximately 10%) than isoprenaline. The beta3-AR antagonist L-748,337, beta1-/beta2-AR antagonist nadolol, and beta1-/beta2-/beta3-AR antagonist bupranolol were used to confirm the involvement of beta3-ARs (and not beta1-/beta2-ARs) in these effects. The beta3-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked I(Ca,L) stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, beta3-ARs are positively coupled to L-type Ca2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.     

S33138 (N-[4-[2-[(3aS,9bR)-8-cyano-1,3a,4,9b-tetrahydro[1] benzopyrano[3,4-c]pyrrol-2(3H)-yl)-ethyl]phenyl-acetamide), a preferential dopamine D3 versus D2 receptor antagonist and potential antipsychotic agent: III. Actions in models of therapeutic activity and induction of side effects

In contrast to clinically available antipsychotics, the novel benzopyranopyrrolidine derivative, S33138 (N-[4-[2-[(3aS,9bR)-8-cyano-1,3a,4,9b-tetrahydro[1]benzopyrano[3,4-c]pyrrol-2(3H)-yl)-ethyl]phenyl-acetamide), behaves as a preferential antagonist of D(3) versus D(2) receptors and does not interact with histamine H(1) and muscarinic receptors. In contrast to haloperidol, clozapine, olanzapine, and risperidone, S33138 (0.16-2.5 mg/kg s.c.) did not disrupt performance in passive-avoidance and five-choice serial reaction time procedures. Furthermore, upon either systemic administration (0.04-2.5 mg/kg s.c.) or introduction into the frontal cortex (0.04-0.63 mug/side), S33138 potently attenuated the perturbation of social recognition by scopolamine or a prolonged intersession delay. Over a comparable and low-dose range, S33138 (0.04-0.63 mg/kg s.c.) elevated dialysis levels of acetylcholine in the frontal cortex of freely moving rats. At higher doses (2.5-10.0 mg/kg s.c.), S33138 also increased frontocortical levels of histamine, whereas monoamines, glutamate, glycine, and GABA were unaffected. By analogy to the other antipsychotics, S33138 (0.63-10.0 mg/kg s.c.) inhibited conditioned avoidance responses in rats, apomorphine-induced climbing in mice, and hyperlocomotion elicited by amphetamine, cocaine, dizocilpine, ketamine, and phencyclidine in rats. S33138 (0.16-2.5 mg/kg s.c.) also blocked the reduction of prepulse inhibition elicited by apomorphine. In comparison with the above actions, only "high" doses of S33138 (10.0-40.0 mg/kg s.c.) elicited catalepsy. To summarize, reflecting preferential blockade of D(3) versus D(2) receptors, S33138 preserves and/or enhances cognitive function, increases frontocortical cholinergic transmission, and is active in models of antipsychotic properties at doses well below those inducing catalepsy. In comparison with clinically available agents, S33138 displays, thus, a distinctive and promising profile of potential antipsychotic properties.     

Indinavir trough concentration as a determinant of early nephrolithiasis in HIV-1-infected adults

Indinavir plasma levels are associated with antiretroviral efficacy; however, little data are available regarding toxicity. We assessed the relationship between indinavir pharmacokinetic (PK) characteristics and severe nephrolithiasis as well as other severe or serious adverse reactions. Patients included in the ANRS CO8 APROCO-COPILOTE cohort and receiving 800 mg indinavir three times daily as a first-line protease inhibitor were eligible for this study. To be included in the analysis, their plasma sample at month 1 (M1) had to be available (n = 282) to estimate using population PK modeling, indinavir PK characteristics, ie, maximum (Cmax) and trough plasma (Cres) concentrations, area under the curve (AUC), and observed/predicted concentration ratio (CR). A Cox model was used to estimate the independent effect of Cmax, Cres, AUC, and CR on the hazard of severe nephrolithiasis and serious adverse reactions. At M1, median Cmax was 6205 ng/mL, Cres 631 ng/mL, AUC 24,242 ng . h/mL, and CR 0.6. After a median follow up of 12 months, 11% of patients (30 of 282) had experienced at least one serious adverse reaction among which 12 were nephrolithiasis. In the multivariate analyses, early high indinavir Cres (ie, >/=1000 ng/mL at M1) was associated with a higher rate of severe nephrolithiasis (hazard ratio = 6.7; 95% confidence interval = 1.8-25.2; P < 0.01) and was also associated with a higher rate of all serious adverse reactions but only when nephrolithiasis were included among those cases. Prospective and early indinavir Cres determination should be recommended in the patient's care management and dosage adjustments.