Expression of thymidylate synthase in human cells is an early G1 event regulated by CDK4 and p16INK4A but not E2F

Thymidylate synthase (TS) is the enzyme that catalyses the last step in de novo thymidylate synthesis. It is of interest clinically because it is an effective target for drugs such as 5-fluorouracil, often used in combination therapy. Despite a number of earlier reports indicating that TS is a cell cycle-dependent enzyme, this remains equivocal. Here, we show that in HCT116 cells synchronised by serum starvation, there is a clear dissociation between the expression of cyclin E (a well-characterised cell-cycle protein) and TS. Although both cyclin E and TS mRNA and protein increased during G1, TS upregulation was delayed. Moreover, TS levels did not decrease following S-phase completion while cyclin E decreased sharply. Similarly, clear differences were seen between cyclin E and TS as asynchronously growing HCT116 cells were growth-inhibited by low-serum treatment. In contrast to previous reports using rodent cells, adenovirus-mediated over-expression of E2F1 and cyclin E in three human cell lines had no effect on TS. Cell-cycle progression was blocked by treatment of cells with pharmacological inhibitors of CDK2 and CDK4 and by ectopic expression of p16INK4A. Whereas CDK2 inhibition had no effect on TS levels, inhibition of CDK4 was associated with decreased TS protein levels. These results provide the first evidence that drugs targeting CDK4 may be useful with anti-TS drugs as combination therapy for cancer

Resistance of a Rodent Malaria Parasite to a Thymidylate Synthase Inhibitor Induces an Apoptotic Parasite Death and Imposes a Huge Cost of Fitness

BACKGROUND: The greatest impediment to effective malaria control is drug resistance in Plasmodium falciparum, and thus understanding how resistance impacts on the parasite's fitness and pathogenicity may aid in malaria control strategy. METHODOLOGY/PRINCIPAL FINDINGS: To generate resistance, P. berghei NK65 was subjected to 5-fluoroorotate (FOA, an inhibitor of thymidylate synthase, TS) pressure in mice. After 15 generations of drug pressure, the 2% DT (the delay time for proliferation of parasites to 2% parasitaemia, relative to untreated wild-type controls) reduced from 8 days to 4, equalling the controls. Drug sensitivity studies confirmed that FOA-resistance was stable. During serial passaging in the absence of drug, resistant parasite maintained low growth rates (parasitaemia, 15.5%±2.9, 7 dpi) relative to the wild-type (45.6%±8.4), translating into resistance cost of fitness of 66.0%. The resistant parasite showed an apoptosis-like death, as confirmed by light and transmission electron microscopy and corroborated by oligonucleosomal DNA fragmentation. CONCLUSIONS/SIGNIFICANCE: The resistant parasite was less fit than the wild-type, which implies that in the absence of drug pressure in the field, the wild-type alleles may expand and allow drugs withdrawn due to resistance to be reintroduced. FOA resistance led to depleted dTTP pools, causing thymineless parasite death via apoptosis. This supports the tenet that unicellular eukaryotes, like metazoans, also undergo apoptosis. This is the first report where resistance to a chemical stimulus and not the stimulus itself is shown to induce apoptosis in a unicellular parasite. This finding is relevant in cancer therapy, since thymineless cell death induced by resistance to TS-inhibitors can further be optimized via inhibition of pyrimidine salvage enzymes, thus providing a synergistic impact. We conclude that since apoptosis is a process that can be pharmacologically modulated, the parasite's apoptotic machinery may be exploited as a novel drug target in malaria and other protozoan diseases of medical importance

HRP-4 walks on Soft Feet

The majority of humanoid robots adopt flat feet, a choice that can limit their performance when maneuvering over uneven terrains. Recently, a soft robotic foot designed to adapt to the ground was proposed to overcome part of these limitations. This paper presents the results of testing two such feet on the humanoid robot HRP-4, and compares them to what obtained with the original flat feet of the robot. After describing the SoftFoot and how it has been adapted to the robot, the biped is tested while balancing, stepping and walking. Tests are carried out on flat ground and on obstacles of different heights. For comparison purposes, the original HRP-4 controller has been used for both types of feet with no changes (except for re-evaluation of the CoM position). Analysis of the ankle pitch angle, ankle pitch torque, knee pitch angle, knee pitch torque, waist roll angle and waist pitch angle, show a substantial improvement in obstacle negotiation performance of HRP-4, when using the SoftFoot, even without optimizing the controller to exploit the SoftFoot features

Creating Personalized Dynamic Models

International audienceIn human motion science, the dynamics plays an important role. It relates the movement of the human to the forces necessary to achieve this movement. It also relates the human and its environment through interaction forces. Estimating subject-specific dynamic models is a challenging problem, due to the need for both accurate measurement and modeling formalisms. In the past decade, we have developed solutions for the computation of the dynamic quantities of humans, based on individual (subject specific) models, inspired largely by Robotics geometric and dynamic calibration. In this chapter, we will present the state of the art and our latest advances in this area and show examples of applications to both humans and humanoid robots. With these research results we hope to contribute beyond the field of robotics to the fields of biomechanics and ergonomics, by providing accurate dynamic models of beings