Fatty Acid Profiles of Leishmania major Derived from Human and Rodent Hosts in Endemic Cutaneous Leishmaniasis Areas of Tunisia and Algeria

Leishmaniasis is a protozoal vector-borne disease that affects both humans and animals. In the Mediterranean Basin, the primary reservoir hosts of Leishmania spp. are mainly rodents and canids. Lipidomic approaches have allowed scientists to establish Leishmania spp. lipid profiles for the identification of cell stage specific biomarkers, drug mechanisms of action, and host immune response. Using an in silico approach of global network interaction between genes involved in fatty acid (FA) synthesis followed by the GC-MS approach, we were able to characterize the fatty acid profiles of L. major derived from human and rodent hosts. Our results revealed that the lipid profile of L. major showed similarities and differences with those already reported for other Leishmania species. Phospholipids are the predominant lipid class. FA composition of rodent parasites was characterized by a lower abundance of the precursor C18:2(n-6). One of the rodent clones, which also expressed the lowest lipid abundance in PL and TAG, was the least sensitive clone to the miltefosine drug and has the lowest infection efficiency. Our findings suggest that the lipid composition variation may explain the response of the parasite toward treatment and their ability to infect their host.

Efficiency of cancer treatments: in silico experiments

Despite the advances in the formulation of different therapies to fight cancer, the design of successful protocols is still a challenging problem. In order to provide some indications on the effectiveness of medical treatments, results from in silico experiments are presented based on a mathematical model comprising two cancer populations competing for resources and with different susceptibilities to the action of therapies. The focus is on the outcome of protocols in which the total dose can be administered with different time distributions. An efficiency index is proposed to quantify the effectiveness of different protocols. Simulations show that a standard dose chemotherapy is effective when the sensitive clone has a marked competitive advantage, whereas its outcome is much worse when a resistant clone emerges; obviously combinations of immune and chemotherapy work better. These results, in accord with previous finding reported in the literature, stress the importance to take into account competitive interactions among cancer clones to decide which therapeutic strategy should be adopted. However, it is not just the efficiency that changes in these different configurations of clonal composition and therapy timing. A general rule seems to emerge: when evolutionary pressures are strong, the best protocols entail and early starting of the treatment, whereas, on the contrary, when interactions among clones are weak, therapy should start later. Finally the model has been adapted to investigate the relative efficiency of different protocols, by using data reported in literature regarding experiments with breast cancer cells.

Liquid biopsy for optimizing the rechallenge of cetuximab in metastatic colorectal cancer: Additional study of E-Rechallenge Trial.

585 Background: Several previous reports indicated that cetuximab (Cmab) rechallenge may be efficacious in some patients for whom Cmab was previously effective. Liquid biopsy can detect the some emerging mutations for resistance with Cmab. Considering the plasticity and elasticity of sensitive clone, we assumed we could identify the patients with benefit from Cmab rechallenge by liquid biopsy. This current study investigates the predictability of efficacy for Cmab rechallenge by liquid biopsy in the E-Rechallenge Trial. Methods: The E-Rechallenge Trial is a multicenter phase II study in mCRC patients who have become refractory to fluoropyrimidines, L-OHP, CPT-11, Cmab, and bevacizumab, and in whom previous treatment with Cmab was effective in any earlier line (achieving CR, PR, or SD that persisted for ≥ 6 months). The other main eligibility criteria are; RAS wild type, measurable disease, aEFI ≥ 16 weeks between the last dose of Cmab during previous treatment and the start of Cmab rechallenge. Protocol treatment is the combination of weekly Cmab with biweekly CPT-11. Additional research of ctDNA was conducted optionally. Baseline plasma samples were analyzed for KRAS, NRAS, BRAF and EGFR S492R mutations using digital PCR (LBx probe, RIKEN GENESIS). A cut-off of the mutation allele frequency was > 0.1%. Results: Between Dec. 2014 and Oct. 2017, 33 patients were enrolled. The primary endpoint; the rates of PR/SD/PD were PR 15.6%/SD 40.6%/PD 43.8%. Twenty-four of 33 patients participated in the additional research. In the additional cohort, the rates of PR/SD/PD were PR 12.5%/SD 50.0%/PD 37.5%. The mutations were detected at the baseline of Cmab rechallenge as followings; KRAS exon 2 29.2%, exon 3,4 33.3%, BRAF V600E 12.5%, EGFR S492R 12.5%. In wild type of these genes the PR and SD rate increased to 25% and 50%, respectively. Conclusions: Cmab rechallenge showed some activity in the salvage setting, in patients for whom Cmab was previously effective. KRAS, BRAF, and EGFR S492R screening by liquid biopsy may contribute to identify patients with benefit from Cmab rechallenge. The additional data of ctDNA may be provided in the conference. Clinical trial information: UMIN 000016439.

Growth and Ethylene Production in Eucalyptus Clones Sensitive to Shoot Blight, Submitted to Hypoxia and High Levels of Manganese

Eucalyptus shoot blight in the Rio Doce Valley (ESBVRD) is a physiological anomaly that has been related to hypoxic environment and Mn excess. This study had the objective of understanding the mechanisms involved in the differential tolerance of eucalyptus clones to ESBVRD. Two experiments were carried out: (I) two clones, a sensitive and a ESBVRD tolerant, two O2 concentrations (normal and hypoxic) and five concentrations of Mn (0, 5, to 90 mg L-1); (II) same two eucalyptus clones, two O2 concentrations (normal and mild hypoxic) and two Mn concentrations (30 and 300 mg L-1). The hypoxic condition reduced the plant dry weight and increased the production of ethylene in the two clones, both more strikingly in the sensitive clone. The O2 deficiency was the first factor predisposing the clones to ESBVRD, in contrast to Mn excess, that appeared to act over a longer period for the expression of the symptoms.

Evolutionary Patterns of Cytogenetically Normal Acute Myeloid Leukemia Correlate with Time to Relapse

Even though two-thirds of acute myeloid leukemia (AML) patients respond to induction chemotherapy and achieve complete remission (CR), the majority of these patients will eventually relapse. The time from CR to relapse is an important clinical indicator of disease aggressiveness, as patients relapsing within the first 6 months after initial diagnosis have a poorer prognosis in terms of response to salvage therapy and overall survival compared to patients with a later relapse. To learn about the evolution during the course of disease, we analyzed the somatic mutation patterns from initial diagnosis to relapse in 50 cytogenetically normal (CN) AML patients. Based on the ELN classification, 38% of the patients (n=19) were assigned as "favorable" at diagnosis, all other patients were classified as "intermediate-I". ELN classification was associated with time to relapse as "intermediate-I" patients relapsed earlier than "favorable" patients (median 9.3 months vs. 16.1 months, p=0.008, log-rank test). Somatic alterations were detected by exome sequencing and confirmed by targeted amplicon sequencing of matched diagnostic, remission and relapse samples. FLT3-ITD and NPM1 mutation status were obtained from routine diagnostic tests as the reliable detection of these markers by NGS remains challenging. The vast majority of somatic alterations were present both at diagnosis and at relapse, hereafter referred to as stable mutations (70%, Fig. 1A). All patients in our cohort had ≥1 stable mutation with DNMT3A being the most stably altered gene. In 47 out of 50 patients (94%), we observed mutations that were only found at diagnosis or only at relapse. Based on the mutation patterns, four distinct 'evolutionary' subgroups of patients were defined (Fig. 1B): (I) patients with an identical mutation profile at diagnosis and at relapse ("stable", n=3, 6%), (II) patients who gained mutations at relapse ("stable + gain", n=24, 48%), (III) patients that lost mutations at relapse ("stable + loss", n=8, 16%), and (IV) patients with both loss and gain of mutations at relapse ("mixed", n=15, 30%). Mutations that were lost during the course of the disease were detected in e.g. PTPN11 or NRAS. Relapse-specific mutations were identified in e.g. IDH1/2, WT1, KPNB1 or KDM6A. Evolutionary subgroups showed differences in time to relapse (Fig. 1C). Patients with "stable + loss" relapsed earlier (median 4.1 months) than patients with gain of mutation at relapse (groups "stable + gain" and "mixed", median 12.2 months). All patients in the category "stable + loss" developed relapse within the first year after complete remission. The "stable" group of 3 patients showed an intermediate time to relapse (median 9.6 months), but was too small for a statistically valid comparison. Ultimately, the genetic evolution of CN-AML patients without gain of new mutations at relapse (categories "stable" and "stable + loss") was associated with significantly earlier relapse compared to patients that gained mutations at relapse (categories "stable + gain" and "mixed", Fig. 1D, p=0.001, log-rank test). Distinct predominant patterns of clonal evolution were observed in the ELN genetic groups, as only one patient of the "stable + loss" group was initially classified as "favorable". Interestingly, applying the ELN classification on relapse samples revealed a switch from "favorable" to "intermediate-I" in six patients, all with gain of mutations at relapse. This points towards more aggressive genetic profiles at relapse in these patients. The acquisition of mutations and/or the outgrowth of a resistant clone during/after chemotherapy might require a longer time or is per se associated with a longer time to relapse and a more favorable prognosis. Loss of mutations at relapse suggest the presence of two clones at diagnosis, with a chemotherapy resistant clone expanding after the eradication of a chemotherapy sensitive clone. As both clones share mutations and only the sensitive clone contains specific alterations, the resistant clone might be an ancestor of the sensitive clone. Taken together, in some patients the AML cells may require additional genetic alterations to become chemotherapy resistant, whereas in other patients the selective eradication of a sensitive clone is a potential mechanism underlying disease progression. Understanding the evolution of AML under selective pressure of chemotherapy is essential to cure or prevent AML relapse. Disclosures Hiddemann: Roche: Other: Grants; Genentech: Other: Grants; Roche: Membership on an entity's Board of Directors or advisory committees.

Manganese accumulation and its relation to "eucalyptus shoot blight in the Vale do Rio Doce"

Eucalyptus Shoot Blight in the Vale do Rio Doce (ESBVRD) is an anomaly that leads to reduced growth and, in more extreme cases, to death of eucalyptus plants. Initially diagnosed in plantations in the region of the Vale do Rio Doce, in the State of Minas Gerais, Brazil, this problem has also been found in plantations in other regions of the country and even in other countries. Although the symptoms of this anomaly are well-known, its causes are not yet understood. The aim of this study was to evaluate the cause-effect relationship between accumulation of manganese (Mn) in eucalyptus clones and ESBVRD. Characterization of the environment in areas of greater occurrence of this problem in regard to soil, climate and fluctuation of the water table was undertaken in eucalyptus plantations of the Celulose Nipo-brasileira S.A. (Cenibra) company in the region of the Vale do Rio Doce. Plant tissues were sampled in two situations. In the first situation, diagnosis occurred in the initial phase of the anomaly in clones with differentiated tolerance to the problem; in the second situation, diagnosis was made in a single clone, considered to be sensitive, in two time periods - in the phase with the strong presence of symptoms and in the recovery phase, in areas of occurrence and in areas of escape from the problem. The most ESBVRD-sensitive clone showed much higher (4.8 times higher) leaf Mn contents than more tolerant clones. In plants with the anomaly, Mn leaf contents were greater than 3,070 mg kg-1, much greater than the quantity found in those without the anomaly (734 mg kg-1). In the period in which the symptoms began to wane, there was a sharp decline in leaf Mn contents, from 2,194 to 847 mg kg-1. Manganese content in the above ground part and plant litter (44.4 g ha-1) in the area of occurrence of the anomaly was three times greater than that found in these same components (14.1 g ha-1) in the area of absence of the symptom. Based on the evidence found, such as the existence of environmental conditions favorable to high Mn availability to the plants in the areas of greatest incidence of ESBVRD, greater uptake of Mn in sensitive clones and in plants with symptoms, and a synchronism between the intensity of symptoms of ESBVRD and leaf Mn contents, it may be inferred that temporary excess of Mn in eucalyptus plants is closely related to ESBVRD.

Reproductive performance of asexual clones of the peach-potato aphid, (Myzus persicae, Homoptera: Aphididae), colonising Scotland in relation to host plant and field ecology

The population of peach-potato aphid, Myzus persicae (Sulzer), in Scotland comprises large numbers of a few superclones with much smaller numbers of other clones, and the reason for their differential success has yet to be elucidated. In the current study, the reproduction of lineages derived from these clones was measured by counting the numbers of offspring produced by a one-day-old nymph after 15 days. This was measured on four plant species, including local agricultural hosts and at two different temperatures (14 and 18°C). There were significant differences in clonal lineage reproduction on different hosts and at different temperatures and amongst clonal lineages on the same hosts at the same temperature. Lineages of local insecticide sensitive clones did not have the best reproductive potential; instead, a recently introduced clonal lineage carrying MACE insecticide resistance was the best reproducer. The clonal lineage with the lowest reproductive potential also carried insecticide resistance, but this was kdr. A lineage from a local insecticide-sensitive clone was the least affected by reduced temperature. There was evidence of host plant specialisation in some of the clonal lineages.

Synthesis and Antimalarial Activities of Cyclen 4-Aminoquinoline Analogs

ABSTRACT In an attempt to augment the efficacy of 7-chloro 4-aminoquinoline analogs and also to overcome resistance to antimalarial agents, we synthesized three cyclen (1,4,7,10-tetraazacyclododecane) analogs of chloroquine [a bisquinoline derivative, 7-chloro-4-(1,4,7,10-tetraaza-cyclododec-1-yl)-quinoline HBr, and a 7-chloro-4-(1,4,7,10-tetraaza-cyclododec-1-yl)-quinoline-Zn2+ complex]. The bisquinoline displays the most potent in vitro and in vivo antimalarial activities. It displays 50% inhibitory concentrations (IC50s) of 7.5 nM against the D6 (chloroquine-sensitive) clone of Plasmodium falciparum and 19.2 nM against the W2 (chloroquine-resistant) clone, which are comparable to those of artemisinin (10.6 and 5.0 nM, respectively) and lower than those of chloroquine (10.7 and 87.2 nM, respectively), without any evidence of cytotoxicity to mammalian cells, indicating a high selectivity index (>1,333 against D6 clone and >521 against W2 clone). Potent antimalarial activities of the bisquinoline against chloroquine- and mefloquine-resistant strains of P. falciparum were also confirmed by in vitro [3H]hypoxanthine incorporation assay. The in vivo antimalarial activity of the bisquinoline, as determined in P. berghei-infected mice, is comparable to that of chloroquine (50% effective dose, ≤1.1 mg/kg when given orally); no apparent toxicity has been observed up to the highest dose tested (3 × 30 mg/kg). The bisquinoline inhibits in vitro hemozoin (β-hematin) formation with an IC50 of 1.1 μM, which is about 10-fold more potent than chloroquine (IC50 9.5 μM). Overall, this article describes the discovery of a new class of cyclen 4-aminoquinoline analogs as potent antimalarial drugs.

Synthesis, Antimalarial Activity, and Intracellular Targets of MEFAS, a New Hybrid Compound Derived from Mefloquine and Artesunate

ABSTRACT A new synthetic antimalarial drug, a salt derived from two antimalarial molecules, mefloquine (MQ) and artesunate (AS), here named MEFAS, has been tested for its pharmacological activity. Combinations of AS plus MQ hydrochloride are currently being used in areas with drug-resistant Plasmodium falciparum parasites; although AS clears parasitemia in shorter time periods than any other antimalarial drug, it does not cure infected patients; in addition, MQ causes side effects and is rather expensive, important problems considering that malaria affects mostly populations in poor countries. Here, we show that MEFAS is more effective than the combination of AS and MQ, tested in parallel at different mass proportions, against P. falciparum (chloroquine-resistant clone W2 and chloroquine-sensitive clone 3D7) in vitro and in mice infected with Plasmodium berghei, promoting cure of this infection. MEFAS tested against HepG2 hepatoma cells exhibited lower toxicity than the antimalarials AS and MQ alone or combined. Possible targets of MEFAS have been studied by confocal microscopy using fluorescent probes (Fluo-4 AM and BCECF-AM) in P. falciparum synchronous culture of W2-infected red blood cells. Dynamic images show that MEFAS exhibited intracellular action increasing cytoplasmic Ca2+ at 1.0 ng/ml. This effect was also observed in the presence of tapsigargin, an inhibitor of SERCA, suggesting an intracellular target distinct from the endoplasmic reticulum. Trophozoites loaded with BCECF-AM, when treated with MEFAS, were still able to mobilize protons from the digestive vacuole (DV), altering the pH gradient. However, in the presence of bafilomycin A1, an inhibitor of the H+ pump from acidic compartments of eukaryotic cells, MEFAS had no action on the DV. In conclusion, the endoplasmic reticulum and DV are intracellular targets for MEFAS in Plasmodium sp., suggesting two modes of action of this new salt. Our data support MEFAS as a candidate for treating human malaria.